EP1032257A1

EP1032257A1 - MONOTERPENE SYNTHASES FROM GRAND FIR ($i(ABIES GRANDIS))

Info

Publication number: EP1032257A1
Application number: EP98935641A
Authority: EP
Inventors: Joerg Bohlmann; Christopher L. Steele; Rodney B. Croteau
Original assignee: University of Washington; Washington State University Research Foundation
Current assignee: University of Washington; Washington State University Research Foundation
Priority date: 1997-07-11
Filing date: 1998-07-10
Publication date: 2000-09-06
Also published as: EP1032257A4; TW585918B; CA2296664A1; AU8483998A; WO1999002030A1

Abstract

cDNAs encoding myrcene synthase, (-)-limonene synthase and (-)-pinene synthase from Grand fir (Abies grandis) haven been isolated and sequenced, and the corresponding amino acid sequences have been determined. Accordingly, isolated DNA sequences (SEQ ID NO:1; SEQ ID NO:3 and SEQ ID NO:5) are provided which code for the expression of myrcene synthase (SEQ ID NO:2), (-)-pinene synthase (SEQ ID NO:4) and (-)-limonene synthase (SEQ ID NO:6) , respectively, from Grand fir (Abies grandis). In other aspects, replicable recombinant cloning vehicles are provided which code for myrcene synthase, (-)-limonene synthase and (-)-pinene synthase, or for a base sequence sufficiently complementary to at least a portion of myrcene synthase, (-)-limonene synthase or (-)-pinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding myrcene synthase, (-)-limonene synthase or (-)-pinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant myrcene synthase, (-)-limonene synthase or (-)-pinene synthase may be used to obtain expression or enhanced expression of myrcene synthase, (-)-limonene synthase or (-)-pinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of myrcene synthase, (-)-limonene synthase and (-)-pinene synthase, or the production of their products.

Description

„,„„„,„ 9/02030

MONOTERPENE SYNTHASES FROM GRAND FIR (Abies grandis)

Field of the Invention The present invention relates to nucleic acid sequences which code for monoterpene synthases from gymnosperm plant species, in particular from Grand fir (Abies grandis), including limonene synthase, myrcene synthase, and pinene synthase, to vectors containing the sequences, to host cells containing the sequences, to plant seeds expressing the sequences and to methods of producing recombinant monoterpene synthases and their mutants. Background of the Invention

Chemical defense of conifer trees against bark beetles and their associated fungal pathogens relies primarily upon constitutive and inducible oleoresin biosynthesis (Johnson, M.A., and Croteau, R. (1987) in Ecology and Metabolism of Plant Lipids (Fuller, G., and Nes, W.D., eds.) pp. 76-91, American Chemical Society Symposium Series 325, Washington, DC; Gijzen, M., Levvinsohn, E., Savage, T.J., and Croteau, R.B. (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., Buttery, R.G., and Sugisawa, H., eds.) pp. 8-22, American Chemical Society Symposium Series 525, Washington, DC). This defensive secretion is a complex mixture of monoterpene and sesquiterpene olefins (turpentine) and diterpene resin acids (rosin) that is synthesized constitutively in the epithelial cells of specialized structures, such as resin ducts and blisters or, in the case of induced oleoresin formation, in undifferentiated cells surrounding wound sites (Lewinsohn, E., Gijzen, M., Savage, T.J., and Croteau, R. (1991) Plant Physiol 96:38-43). The volatile fraction of conifer oleoresin, which is toxic to both bark beetles and their fungal associates (Raffa, K.F., Berryman, A.A., Simasko, J., Teal, W., and Wong, B.L. (1985) Environ. Entomol 14:552-556), may consist of up to 30 different monoterpenes (Lewinsohn, E., Savage, T.J., Gijzen, M., and Croteau, R. (1993) Phyloche . Anal. 4:220-225), including acyclic types (e.g., myrcene), onocyclic types (e.g., limonene) and bicyclic types (e.g., pinenes) (FIGURE 1). Although the oleoresin is toxic, many bark beetle species nevertheless employ turpentine volatiles in host selection and can convert various monoterpene components into aggregation or sex pheromones to promote coordinated mass attack of the host (Gijzen, M., Lewinsohn, E., Savage, T.J., and Croteau, R.B. (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., Buttery, R.G., and Sugisawa, H., eds.) pp. 8- 22, American Chemical Society Symposium Series 525, Washington, DC; Byers, J.A. (1995) in Chemical Ecology of Insects 2 (Carde, R.T., and Bell, W.J., eds.) pp. 154-213. Chapman and Hall, New York). In Grand fir (Abies grandis), increased formation of oleoresin monoterpenes, sesquiterpenes and diterpenes is induced by bark beetle attack (Lewinsohn, E., Gijzen, M., Savage, T.J., and Croteau, R. (1991) Plant Physiol. 96:38-43; Raffa, K.F., and Berryman, A.A. (1982) Can. Entomol. 114:797-810; Lewinsohn, E., Gijzen, M., and Croteau, R. (1991) Plant Physiol. 96:44-49), and this inducible defense response is mimicked by mechanically wounding sapling stems (Lewinsohn, E., Gijzen, M., Savage, T.J., and Croteau, R. (1991) Plant Physiol. 96:38-43; Lewinsohn, E., Gijzen, M., and Croteau, R. (1991) Plant Physiol. 96:44-49; Funk, C, Lewinsohn, E., Stofer Vogel, B., Steele C, and Croteau, R. (1994) Plant Physiol. 106:999-1005). Therefore, Grand fir has been developed as a model system to study the biochemical and molecular genetic regulation of constitutive and inducible terpene biosynthesis in conifers (Steele, C, Lewinsohn, E., and Croteau, R. (1995) Proc. Nail Acad. Sci. USA 92:4164-4168).

Most monoterpenes are derived from geranyl diphosphate, the ubiquitous C₍o intermediate of the isoprenoid pathway, by synthases which catalyze the divalent metal ion-dependent ionization (to 1, FIGURE 1) and isomerization of this substrate to enzyme-bound linalyl diphosphate which, following rotation about C2-C3, undergoes a second ionization (to 2, FIGURE 1) followed by cyclization to the α-terpinyl cation, the first cyclic intermediate en route to both monocyclic and bicyclic products (Croteau, R., and Cane, D.E. (1985) Methods Enzymol 110:383- 405; Croteau, R. (1987) Chem. Rev. 87:929-954) (FIGURE 1). Acyclic monoterpenes, such as myrcene, may arise by deprotonation of carbocations 1 or 2, whereas the isomerization step to linalyl diphosphate is required in the case of cyclic types, such as limonene and pinenes, which cannot be derived from geranyl diphosphate directly because of the geometric impediment of the trαπs-double bond at C2-C3 (Croteau, R., and Cane, D.E. (1985) Methods Enzymol 110:383-405; Croteau, R. (1987) Chem. Rev. 87:929-954). Many monoterpene synthases catalyze the formation of multiple products, including acyclic, monocyclic and bicyclic types, by variations on this basic mechanism (Gambliel, H., and Croteau, R. (1984) J. Biol. Chem. 259:740-748; Croteau, R., Satterwhite, D.M., Cane, D.E., and Chang, C.C. (1988) J. Biol. Chem. 263:10063-10071; Croteau, R., and Satterwhite, D.M. (1989) J. Biol. Chem. 264:15309-15315). For example, (-)-limonene synthase, the principal monoterpene synthase of spearmint (Mentha spicata) and peppermint (M. x piperita), produces small amounts of myrcene, (-)-α-pinene and (-)-β-pinene in addition to the monocyclic product (Rajaonarivony, J.I.M., Gershenzon, J., and Croteau, R. (1992) Arch. Biochem. Biophys. 296:49-57; Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J.. and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024. Conversely, six diflerent inducible monoterpene synthase activities have been demonstrated in extracts of wounded Grand fir stem (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289:267-273) indicating that formation of acyclic, monocyclic and bicyclic monoterpenes in this species involves several genes encoding distinct catalysts. The inducible (-)-pinene synthase has been purified (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173), and isotopically sensitive branching experiments employed to demonstrate that this enzyme synthesizes both (-)-α- and (-)-β-pinene (Wagschal, K., Savage, T.J., and Croteau, R. (1991) Tetrahedron 47:5933-5944).

Deciphering the molecular genetic control of oleoresinosis and examining structure-function relationships among the monoterpene synthases of Grand fir requires isolation of the cDNA species encoding these key enzymes. Although a protein-based cloning strategy was recently employed to acquire a cDNA for the major wound-inducible dilerpene synthase from Grand fir, abietadiene synthase

(Funk, C, Lewinsohn, E., Stofer Vogel, B., Steele C, and Croteau, R. (1994) Plant Physiol. 106:999- 1005; LaFever, R.E., Stofer Vυgel, B., and Croteau, R. (1994)

Arch. Biochem. Biophys. 313:139-149; Stofer Vogel, B., Wildung, M.R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268), all attempts at the reverse genetic approach to cloning of Grand fir monoterpene synthases have failed (Steele,

C, Lewinsohn, E., and Croteau, R. (1995) Proc. Natl. Acad. Sci. USA 92:4164- 4168). As an alternative, a similarity-based PCR strategy was developed (Steele, C, Lewinsohn, E., and Croteau, R. (1995) Proc. Natl. Acad. Sci. USA 92:4164-4168) that employed sequence information from terpene synthases of angiosperm origin, namely a monoterpene synthase, (-)-45'-limonene synthase, from spearmint (Mentha spicata, Lamiaceae) (Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024), a sesquiterpene synthase, 5-e/? -aristolochene synthase, from tobacco (Nicoliana tabacum, Solanaceae) (Facchini, P.J., and Chappell, J. (1992) Proc. Nail. Acad. Sci. USA 89: 1 1088-1 1092), and a diterpene synthase, casbene synthase, from castor bean (Ricinus communis, Euphorbiaceae) (Mau, C.J.D., and West, CA. (1994) Proc. Natl. Acad. Sci. ?7&4 91:8497-8501).

Monoterpenes have significant potential for cancer prevention and treatment. Monoterpenes such as limonene, perillyl alcohol, carvone, geraniol and farnesol not only reduce tumor incidence and slow tumor proliferation, but have also been reported to cause regression of established solid tumors by initiating apoptosis (Mills J.J., Chari R.S., Boyer I.J., Gould M.N., Jirtle R.L., Cancer Res., 55:979-983, 1995). Terpenes have activity against cancers such as mammary, colon, and prostate. Clinical trials are being pursued (Seachrist L, J. NIH Res. 8:43) in patients with various types of advanced cancers to validate the health benefits of dietary terpenes for humans. However, terpenes are present in Western diets at levels that are probably inadequate for any significant preventive health benefits. Daily supplementation of the diet with a terpene concentrate (10-20 g/day) would appear to be the most rational strategy for dietary therapy of diagnosed cases of cancer. This invention envisages the production of such nutritionally beneficial terpenes in vegetable oils consumed daily via the engineering of relevant genes from Grand fir into oil seed crop plants such as oil seed brassica (canola), soybean and corn.

Brief Description of the Drawings The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE 1 is a schematic representation depicting the mechanism for the conversion of geranyl diphosphate to myrcene, (-)-limonene, β-phellandrene,

(-)-α-pinene and (-)-β-pinene by monoterpene synthases from Grand fir. Formation of the monocyclic and bicyclic products requires preliminary isomerization of geranyl diphosphate to linalyl diphosphate. The acyclic product could be formed from either geranyl diphosphate or linalyl diphosphate via carbocations 1 or 2. OPP denotes the diphosphate moiety.

FIGURE 2 is a sequence comparison of plant terpene synthases. A three- letter designation (Tps) for the gene family is proposed with sub-groups (Tpsa through Tpsf) defined by a minimum of 40 % amino acid identity between members. The numbers in parenthesis are the references to the published sequences.

FIGURE 3 depicts a GLC-MS analysis of the products of the recombinant protein encoded by AG2.2 (SEQ ID NO:l), the sequence of the protein encoded by clone AG2.2 being set forth in SEQ ID NO:2. The GLC profile of the total pentane- soluble products generated from geranyl diphosphate when incubated with a cell-free extract of E. coli XLl-Blue/pGAG2.2 is illustrated (a), as are the mass fragmentation patterns for the monoterpene product with R_j = 12.22 min (b) and for authentic myrcene (c).

FIGURE 4 depicts a GLC-MS analysis of the products of the recombinant protein encoded by AG3.18 (SEQ ID NO:3), the sequence of the protein encoded by clone AG3.18 (SEQ ID NO:3) being set forth in SEQ ID NO:4. The GLC profile of the total pentane-soluble products generated from geranyl diphosphate when incubated with a cell-free extract of E. coli XLl-Blue/pGAG3.18 is illustrated (a), as are the mass fragmentation patterns (selected ion mode) for the monoterpene products with R₍ = 1 1.34 min (b), and R( = 13.37 min (d), and for authentic α-pinene (c) and authentic β-pinene (e).

FIGURE 5 depicts a GLC-MS analysis of the products of the recombinant protein encoded by AGIO (SEQ ID NO:5), the sequence of the protein encoded by clone AGIO (SEQ ID NO:5) being set forth in SEQ ID NO:6. The GLC profile of the total pentane-soluble products generated from geranyl diphosphate when incubated with a cell-free extract of E. coli BL21(DE3)/pSBAG10 is illustrated (a), as are the mass fragmentation patterns for the principal monoterpene product with R_t = 13.93 min (b) and for authentic limonene (c).

Summary of the Invention In accordance with the foregoing, cDNAs encoding myrcene synthase,

(-)-limonene synthase and (-)-pincne synthase from Grand fir (Abies grandis) have been isolated and sequenced, and the corresponding amino acid sequences have been deduced. Accordingly, the present invention relates to isolated DNA sequences which code for the expression of myrcene synthase, such as the sequence designated SEQ ID NO:l which encodes myrcene synthase from Grand fir (Abies grandis), for the expression of (-)-pinene synthase, such as the sequence designated SEQ ID NO:3, which encodes the (-)-pinene synthase from Grand fir (Abies grandis) and for the expression of (-)-limonene synthase, such as the sequence designated SEQ ID NO:5, which encodes (-)-limonene synthase from Grand fir (Abies grandis). In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence, e.g., a DNA sequence which codes for a myrcene synthase, (-)-limonene synthase or (-)-pinene synthase, or for a base sequence sufficiently complementary to at least a portion of DNA or RNA encoding myrcene synthase, (-)-limonene synthase or (-)-pinene synthase to enable hybridization therewith (e.g., antisense RNA or fragments of DNA complementary to a portion of DNA or RNA molecules encoding myrcene synthase, (-)-limonene synthase or (-)-pinene synthase which are useful as polymerase chain reaction primers or as probes for any of the foregoing synthases or related genes). In yet other aspects of the invention, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention. Thus, the present invention provides for the recombinant expression of myrcene synthase, (-)-limonene synthase and (-)-pinene synthase, and the inventive concepts may be used to facilitate the production, isolation and purification of significant quantities of recombinant myrcene synthase, (-)-limonene synthase and (-)-pinene synthase (or of their primary enzyme products) for subsequent use, to obtain expression or enhanced expression of myrcene synthase, (-)-limonene synthase and (-)-pinene synthase in plants, microorganisms or animals, or may be otherwise employed in an environment where the regulation or expression of myrcene synthase, (-)-limυncnc synthase and (-)-pinene synthase is desired for the production of these synthases, or their enzyme products, or derivatives thereof. In another aspect, the present invention relates to manipulation of monoterpene production to enhance resistance to insects and/or accumulate nutritionally beneficial monoterpenes in oil seeds.

Detailed Description of the Preferred Embodiment As used herein, the terms "amino acid" and "amino acids" refer to all naturally occurring L-α-amino acids or their residues. The amino acids are identified by either the single-letter or three-letter designations:

Asp D aspartic acid He I isoleucine

Thr T threonine Leu L leucine Ser S serine Tyr Y tyrosine Glu E glutamic acid Phe F phenylalanine

Pro P proline His H histidine

Gly G glycine Lys K lysine

Ala A alanine Arg R arginine

Cys C cysteine Trp W tryptophan

Val V valine Gin Q glutamine

Met M methionine Asn N asparagine

As used herein, the term "nucleotide" means a monomeric unit of DNA or RNA containing a sugar moiety (pentose), a phosphate and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (V carbon of pentose) and that combination of base and sugar is called a nucleoside. The base characterizes the nucleotide with the four bases of DNA being adenine ("A"), guanine ("G"), cytosine ("C") and thymine ("T"). Inosine ("I") is a synthetic base that can be used to substitute for any of the four, naturally-occurring bases (A, C, G or T). The four RNA bases are A,G,C and uracil ("U"). The nucleotide sequences described herein comprise a linear array of nucleotides connected by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses.

The term "percent identity" means the percentage of amino acids or nucleotides that occupy the same relative position when two amino acid sequences, or two nucleic acid sequences are aligned side by side.

The term "percent similarity" is a statistical measure of the degree of relatedness of two compared protein sequences. The percent similarity is calculated by a computer program that assigns a numerical value to each compared pair of amino acids based on chemical similarity (e.g., whether the compared amino acids are acidic, basic, hydrophobic, aromatic, etc.) and/or evolutionary distance as measured by the minimum number of base pair changes that would be required to convert a codon encoding one member of a pair of compared amino acids to a codon encoding the other member of the pair. Calculations are made after a best fit alignment of the two sequences have been made empirically by iterative comparison of all possible alignments. (Henikoff, S. and Henikoff, J.G., Proc. Nat'l. Acad. Sci.

1/5^ 89: 10915-10919, 1992).

"Oligonucleotide" refers to short length single or double stranded sequences of deoxyribonucleotides linked via phosphodiester bonds. The oligonucleotides are chemically synthesized by known methods and purified, for example, on polyacrylamide gels. The term "myrcene synthase" is used herein to mean an enzyme capable of generating multiple monoterpenes from geranyl diphosphate. The principal and characteristic monoterpene synthesized by myrcene synthase is myrcene, which constitutes at least about 60% of the monoterpene mixture synthesized by myrcene synthase from geranyl diphosphate.

The term "(-)-limonene synthase" is used herein to mean an enzyme capable of generating multiple monoterpenes from geranyl diphosphate. The principal and characteristic monoterpene synthesized by (-)-limonene synthase is (-)-limonene, which constitutes at least about 60% of the monoterpene mixture synthesized by (-)-limonene synthase from geranyl diphosphate.

The term "(-)-pinene synthase" is used herein to mean an enzyme capable of generating multiple monoterpenes from geranyl diphosphate. The principal and characteristic monoterpene synthesized by (-)-pinene synthase is (-)-pinene, which comprises at least about 60% of the monoterpene mixture synthesized by (-)-pinene synthase from geranyl diphosphate.

The abbreviation "SSPE" refers to a buffer used in nucleic acid hybridization solutions. The 20X (twenty times concentrate) stock SSPE buffer solution is prepared as follows: dissolve 175.3 grams of NaCl, 27.6 grams of NaH₂PO₄H₂O and

7.4 grams of EDTA in 800 millilitres of H₂O. Adjust the pH to pH 7.4 with NaOH. Adjust the volume to one liter with H₂O.

The terms "alteration", "amino acid sequence alteration", "variant" and "amino acid sequence variant" refer to monoterpene synthase molecules with some differences in their amino acid sequences as compared to the corresponding, native, i.e., naturally-occurring, monoterpene synthases. Ordinarily, the variants will possess at least about 70% homology with the corresponding native monoterpene synthases, and preferably, they will be at least about 80% homologous with the corresponding, native monoterpene synthases. The amino acid sequence variants of the monoterpene synthases falling within this invention possess substitutions, deletions, and/or insertions at certain positions. Sequence variants of monoterpene synthases may be used to attain desired enhanced or reduced enzymatic activity, modified regiochemistry or stereochemistry, or altered substrate utilization or product distribution.

Substitutional monoterpene synthase variants are those that have at least one amino acid residue in the native monoterpene synthase sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. Substantial changes in the activity of the monoterpene synthase molecules of the present invention may be obtained by substituting an amino acid with a side chain that is significantly different in charge and/or structure from that of the native amino acid. This type of substitution would be expected to affect the structure of the polypeptide backbone and/or the charge or hydrophobicity of the molecule in the area of the substitution.

Moderate changes in the activity of the monoterpene synthase molecules of the present invention would be expected by substituting an amino acid with a side chain that is similar in charge and/or structure to that of the native molecule. This type of substitution, referred to as a conservative substitution, would not be expected tυ substantially alter either the structure of the polypeptide backbone or the charge or hydrophobicity of the molecule in the area of the substitution. Insertional monoterpene synthase variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in the native monoterpene synthase molecule. Immediately adjacent to an amino acid means connected to either the α-carboxy or -amino functional group of the amino acid. The insertion may be one or more amino acids. Ordinarily, the insertion will consist of one or two conservative amino acids. Amino acids similar in charge and/or structure to the amino acids adjacent to the site of insertion are defined as conservative. Alternatively, this invention includes insertion of an amino acid with a charge and/or structure that is substantially different from the amino acids adjacent to the site of insertion. Deletional variants are those where one or more amino acids in the native monoterpene synthase molecules have been removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the monoterpene synthase molecule.

The terms "biological activity", "biologically active", "activity" and "active" refer to the ability of the monoterpene synthases of the present invention to convert geranyl diphosphate to a group of monoterpenes, of which myrcene is the principal and characteristic monoterpene synthesized by myrcene synthase, (-)-limonene is the principal and characteristic monoterpene synthesized by (-)-limonene synthase and (-)-pinene is the principal and characteristic monoterpene synthesized by (-)-pinene synthase. The monoterpenes produced by the monoterpene synthases of the present invention are as measured in an enzyme activity assay, such as the assay described in Example 3. Amino acid sequence variants of the terpene synthases of the present invention may have desirable altered biological activity including, for example, altered reaction kinetics, substrate utilization product distribution or other characteristics such as regiochemistry and stereochemistry.

The terms "DNA sequence encoding", "DNA encoding" and "nucleic acid encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the translated polypeptide chain. The DNA sequence thus codes for the amino acid sequence.

The terms "replicable expression vector" and "expression vector" refer to a piece of DNA, usually double-stranded, which may have inserted into it a piece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of or coincidental with the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA may be generated. In addition, the vector contains the necessary elements that permit translating the foreign DNA into a polypeptide. Many molecules of the polypeptide encoded by the foreign DNA can thus be rapidly synthesized.

The terms "transformed host cell," "transformed" and "transformation" refer to the introduction of DNA into a cell. The cell is termed a "host cell", and it may be a prokaryotic or a eukaryotic cell. Typical prokaryotic host cells include various strains of E. coli. Typical eukaryotic host cells are plant cells, such as maize cells, yeast cells, insect cells or animal cells. The introduced DNA is usually in the form of a vector containing an inserted piece of DNA. The introduced DNA sequence may be from the same species as the host cell or from a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign DNA and some DNA derived from the host species. The following abbreviations are used herein: bp(s), base pair(s); DEAE,

O-diethylaminoethyl; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; GC, gas chromatography; 1PTG, isopropyl-β-D-thiogalactopyranoside; LB, Luria- Bertani; Mopso, 3-(N-morpholino)-2-hydroxypropane-sulfonic acid; MS, mass spectrum/ spectrometry; nt(s), nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction; PVDF, polyvinylidenedifluoride; SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethyl) aminomethane; UV, ultraviolet.

In accordance with the present invention, cDNAs encoding myrcene synthase (SEQ ID NO:l), (-)-pinene synthase (SEQ ID NO:3) and (-)-limonene synthase (SEQ ID NO:5) from Grand fir (Abies grandis) were isolated and sequenced in the following manner. Based on comparison of sequences of limonene synthase from spearmint (Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J., and Croteau, R. (1993) J. Biol Chem. 268:23016-23024), 5-e/?/-aristolochene synthase from tobacco (Facchini, P.J., and Chappell, J. (1992) Proc. Natl. Acad. Sci. rø 89:1 1088-11092), and casbene synthase from castor bean (Mau, C.J.D., and West, CA. (1 94) Proc. Nail. Acad. Sci. USA 91:8497-8501), four conserved regions were identified for which a set of consensus, degenerate primers were synthesized: Primer A (SEQ ID NO:7), Primer B (SEQ ID NO:8), Primer C (SEQ ID NO:9) and Primer D (SEQ ID NO: 10). Primers A (SEQ ID NO:7), B (SEQ ID NO:8), and D (SEQ ID NO: 10) were sense primers, while Primer C (SEQ ID NO:9), was an antisense primer. Each of the sense primers, A (SEQ ID NO:7), B (SEQ ID NO:8) and D (SEQ ID NO: 10), was used for PCR in combination with antisense primer C (SEQ ID NO:9) by employing a broad range of amplification conditions. Analysis of the PCR reaction products by agarose gel electrophoresis revealed that only the combination of primers C (SEQ ID NO:9) and D (SEQ ID NO: 10) generated a specific PCR product of approximately 110 bps.

The 1 10 bps PCR product was gel purified, ligated into a plasmid, and transformed into E. coli XL 1 -Blue cells. Plasmid DNA was prepared from 41 individual transformants and the inserts were sequenced. Four different insert sequences were identified, and were designated as probes 1 (SEQ ID NO:l 1), 2 (SEQ ID NO: 12), 4 (SEQ ID NO:13) and 5 (SEQ ID NO: 14). Probes 1 (SEQ ID NO: 11), 2 (SEQ ID NO:12), 4 (SEQ ID NO:13) and 5 (SEQ ID NO:14) were used to screen a cDNA library made from mRNA extracted from wounded Grand fir stems, and the longest clone that hybridized to each of these probes was isolated and sequenced. Thus, clone AG1.28 (SEQ ID NO: 15) is the longest cDNA clone that hybridized to probe 1 (SEQ ID NO:l l), clone AG2.2 (SEQ ID NO:l) is the longest cDNA clone that hybridized to probe 2 (SEQ ID NO: 12), clone AG4.30 (SEQ ID NO: 17) is the longest cDNA clone that hybridized to probe 4 (SEQ ID NO: 13), and clone AG5.9 (SEQ ID NO: 19) is the longest cDNA clone that hybridized to probe 5 (SEQ ID NO: 14). Truncated clone AG1.28 (SEQ ID NO:15) resembled most closely in size and sequence (72% similarity, 49% identity) a diterpene cyclase, abietadiene synthase, from Grand fir. Clones AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19) encode sesquiterpene synthases. Sequence and functional analysis of clone AG2.2 (SEQ ID NO: l) revealed that it encoded the monoterpene synthase, myrcene synthase.

Alignment of the four new terpene synthase cDNA sequences AG1.28 (SEQ

ID NO:15), AG2.2 (SEQ ID NO: l), AG4.30 (SEQ ID NO:17) and AG5.9 (SEQ ID

NO: 19) with that for abietadiene synthase (Stofer Vogel, B., Wildung, M.R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268) allowed the identification of several, conserved sequence motifs. Two new sense PCR primers, primer E (SEQ ID NO:21) and primer F (SEQ ID NO:22) were designed based on the sequence of the conserved protein sequence motifs. A new antisense PCR primer, primer G (SEQ ID NO:23), was designed based on limited sequence information available from pinene synthase. The combination of primer E (SEQ ID

NO:21) and primer G (SEQ ID NO:23) amplified a cDNA product of 1022 bps, which was designated as probe 3 (SEQ ID NO:24).

Probe 3 (SEQ ID NO:24) was used to screen a cDNA library made from mRNA extracted from wounded Grand fir stems. Hybridization of 10 Grand fir λZAP II cDNA clones with probe 3 (SEQ ID NO:24) yielded two types of signals comprised of about 400 strongly positive clones and an equal number of weak positives, indicating that the probe recognized more than one type of cDNA. Thirty- four of the former clones and eighteen of the latter were purified, the inserts were selected by size (2.0-2.5 kb), and the in vivo excised clones were partially sequenced from both ends. Those clones which afforded weak hybridization signals were shown to contain inserts that were either identical to myrcene synthase clone AG2.2 (SEQ ID NO:l) or exhibited no significant sequence similarity to terpene synthases.

Clones which gave strong hybridization signals segregated into distinct sequence groups represented by clone AG3.18 (SEQ ID NO:3) and clone AGIO (SEQ ID NO:5). Both AG3.18 (SEQ ID NO:3) and AGIO (SEQ ID NO:5) were subcloned into plasmid expression vectors and expressed in E. coli. When extracts of the induced cells were tested for terpene synthase activity with all of the potential prenyl diphosphate substrates, only geranyl diphosphate was utilized. Extracts from E. coli containing the AGIO (SEQ ID NO:5) expression construct converted geranyl diphosphate to the -4S enantiomer of limonene as the major product, indicating that AGIO (SEQ ID NO: 5) encodes (-)-limonene synthase. Similar analysis of the monoterpene products generated from geranyl diphosphate by cell-free extracts of E. coli containing the AG3.18 (SEQ ID NO:3) insert ligated into an expression vector revealed the presence of a 42:58%) mixture of α-pinene and β-pinene, the same product ratio previously described for the purified, native (-)-pinene synthase from Grand fir. These data indicate that AG3.18 (SEQ ID NO:3) encodes (-)-pinene synthase.

The isolation of cDNAs encoding (-)-limonene synthase, (-)-pinene synthase and myrcene synthase permits the development of efficient expression systems for these functional enzymes; provides useful tools for examining the developmental regulation of monoterpene biosynthesis; permits investigation of the reaction mechanism(s) of these unusual, multiproduct enzymes, and permits the isolation of other (-)-limonene synthase, (-)-pinene synthase and myrcene synthases. The isolation of the (-)-limonene synthase, (-)-pinene synthase and myrcene synthase cDNAs also permits the transformation of a wide range of organisms in order to introduce monoterpene biosynthesis de novo, or to modify endogenous monoterpene biosynthesis.

Although the (-)-limonene synthase, (-)-pinene synthase and myrcene synthase proteins set forth in SEQ ID NO:6, SEQ ID NO:4 and SEQ ID NO:2, respectively, direct the enzymes to plastids, substitution of the presumptive targeting sequence of each of these enzymes (SEQ ID NO:2, amino acids 1 to 61 ; SEQ ID NO:4, amino acids 1 to 61 ; SEQ ID NO:6, amino acids 1 to 66) with other transport sequences well known in the art (see, e.g., von Heijne et al., Eur. J. Biochem. 180:535-545, 1989; Stryer, Biochemistry, W.H. Freeman and Company, New York, NY, p. 769 [1988]) may be employed to direct the (-)-limonene synthase, (-)-pinene synthase and myrcene synthase to other cellular or extracellular locations.

In addition to the native (-)-limonene synthase, (-)-pinene synthase and myrcene synthase amino acid sequences of SEQ ID NO:6, SEQ ID NO:4 and SEQ ID NO:2, respectively, sequence variants produced by deletions, substitutions, mutations and/or insertions are intended to be within the scope of the invention except insofar as limited by the prior art. The (-)-limonene synthase, (-)-pinene synthase and myrcene synthase amino acid sequence variants of this invention may be constructed by mutating the DNA sequences that encode the wild-type synthases, such as by using techniques commonly referred to as site-directed mutagenesis. Nucleic acid molecules encoding the monoterpene synthases of the present invention can be mutated by a variety of PCR techniques well known to one of ordinary skill in the art. See, e.g., "PCR Strategies", M.A. Innis, D.H. Gelfand and J.J. Sninsky, eds., 1995, Academic Press, San Diego, CA (Chapter 14); "PCR Protocols: A Guide to Methods and Applications", M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White, eds., Academic Press, NY ( 1990).

By way of non-limiting example, the two primer system utilized in the Transformer Site-Directed Mutagenesis kit from Clontech, may be employed for introducing site-directed mutants into the monoterpene synthase genes of the present invention. Following denaturation of the target plasmid in this system, two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site-directed mutation, the other contains a mutation al another point in the plasmid resulting in elimination of a restriction site. Second strand synthesis is then carried out, lightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli. Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli. This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids. The tight linkage of the two mutations and the subsequent linearization of unmutated plasmids results in high mutation efficiency and allows minimal screening. Following synthesis of the initial restriction site primer, this method requires the use of only one new primer type per mutation site. Rather than prepare each positional mutant separately, a set of "designed degenerate" oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously. Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be restricted and analyzed by electrophoresis on Mutation Detection Enhancement gel (J.T. Baker) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control). The verified mutant duplexes in the pET (or other) overexpression vector can be employed to transform E. coli such as strain E. coli BL21(DE3)pLysS, for high level production of the mutant protein, and purification by standard protocols. The method of FAB-MS mapping can be employed to rapidly check the fidelity of mutant expression. This technique provides for sequencing segments throughout the whole protein and provides the necessary confidence in the sequence assignment. In a mapping experiment of this type, protein is digested with a protease (the choice will depend on the specific region to be modified since this segment is of prime interest and the remaining map should be identical to the map of unmutagenized protein). The set of cleavage fragments is fractionated by microbore HPLC (reversed phase or ion exchange, again depending on the specific region to be modified) to provide several peptides in each fraction, and the molecular weights of the peptides are determined by FAB-MS. The masses are then compared to the molecular weights of peptides expected from the digestion of the predicted sequence, and the correctness of the sequence quickly ascertained. Since this mutagenesis approach to protein modification is directed, sequencing of the altered peptide should not be necessary if the MS agrees with prediction. If necessary to verify a changed residue, CAD-tandem MS/MS can be employed to sequence the peptides of the mixture in question, or the target peptide purified for subtractive Edman degradation or carboxypeptidase Y digestion depending on the location of the modification. In the design of a particular site directed mutagenesis, it is generally desirable to first make a non-conservative substitution (e.g., Ala for Cys, His or Glu) and determine if activity is greatly impaired as a consequence. The properties of the mutagenized protein are then examined with particular attention to the kinetic parameters of K_m and k_cat as sensitive indicators of altered function, from which changes in binding and/or catalysis per se may be deduced by comparison to the native enzyme. If the residue is by this means demonstrated to be important by activity impairment, or knockout, then conservative substitutions can be made, such as Asp for Glu to alter side chain length, Ser for Cys, or Arg for His. For hydrophobic segments, it is largely size that is usefully altered, although aromatics can also be substituted for alkyl side chains. Changes in the normal product distribution can indicate which step(s) of the reaction sequence have been altered by the mutation. Modification of the hydrophobic pocket can be employed to change binding conformations for substrates and result in altered regiochemistry and/or stereochemistry. Other site directed mutagenesis techniques may also be employed with the nucleotide sequences of the invention. For example, restriction endonuclease digestion of DNA followed by ligation may be used to generate deletion variants of (-)-limonene synthase, (-)-pinene synthase and myrcene synthase, as described in section 15.3 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York, NY [1989]). A similar strategy may be used to construct insertion variants, as described in section 15.3 of Sambrook et al., supra.

Oligonucleotide-directed mutagenesis may also be employed for preparing substitution variants of this invention. It may also be used to conveniently prepare the deletion and insertion variants of this invention. This technique is well known in the art as described by Adelman et al. (DNA 2: 183 [1983]); Sambrook et al., supra; "Current Protocols in Molecular Biology", 1991, Wiley (NY), F.T. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.D. Seidman, J.A. Smith and K. Struhl, eds.

Generally, oligonucleotides of at least 25 nucleotides in length are used to insert, delete or substitute two or more nucleotides in the (-)-limonene synthase, (-)-pinene synthase and myrcene synthase molecule. An optimal oligonucleotide will have 12 to 15 perfectly matched nucleotides on either side of the nucleotides coding for the mutation. To mutagenize wild-type (-)-limonene synthase, (-)-pinene synthase and myrcene synthase, the oligonucleotide is annealed to the single-stranded DNA template molecule under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of E. coli DNA polymerase I, is then added. This enzyme uses the oligonucleotide as a primer to complete the synthesis of the mutation-bearing strand of DNA. Thus, a heteroduplex molecule is formed such that one strand of DNA encodes the wild-type synthase inserted in the vector, and the second strand of DNA encodes the mutated form of the synthase inserted into the same vector. This heteroduplex molecule is then transformed into a suitable host cell.

Mutants with more than one amino acid substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If however, the amino acids are located some distance from each other (separated by more than ten amino acids, for example) it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions. An alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: wild-type (-)-limonene synthase, (-)-pinene synthase and myrcene synthase DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on. A gene encoding (-)-limonene synthase, (-)-pinene synthase and myrcene synthase may be incorporated into any organism (intact plant, animal, microbe, etc.), or cell culture derived therefrom, that produces geranyl diphosphate. A (-)-limonene synthase, (-)-pinene synthase and myrcene synthase gene may be introduced into any organism for a variety of purposes including, but not limited to: production of (-)-limonene synthase, (-)-pinene synthase and myrcene synthase, or their products; production or modification of flavor and aroma properties; improvement of defense capability, and the alteration of other ecological interactions mediated by myrcene, (-)-limonene, (-)-pinene, or their derivatives.

Eukaryotic expression systems may be utilized for the production of (-)-limonene synthase, (-)-pinene synthase and myrcene synthase since they are capable of carrying out any required posttranslational modifications and of directing the enzymes to the proper membrane location. A representative eukaryotic expression system for this purpose uses the recombinant baculovirus, Autographa califomica nuclear polyhedrosis virus (AcNPV; M.D. Summers and G.E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures [1986]; Luckow et al., Bio-technology 6:47-55 [1987]) for expression of the terpenoid synthases of the invention. Infection of insect cells (such as cells of the species Spodoptera frugiperda) with the recombinant baculoviruses allows for the production of large amounts of the monoterpenoid synthase proteins. In addition, the baculovirus system has other important advantages for the production of recombinant (-)-limonene synthase, (-)-pinene synthase and myrcene synthase. For example, baculoviruses do not infect humans and can therefore be safely handled in large quantities. In the baculovirus system, a DNA construct is prepared including a DNA segment encoding (-)-limonene synthase, (-)-pinene synthase and myrcene synthase and a vector. The vector may comprise the polyhedron gene promoter region of a baculovirus, the baculovirus flanking sequences necessary for proper cross-over during recombination (the flanking sequences comprise about 200-300 base pairs adjacent to the promoter sequence) and a bacterial origin of replication which permits the construct to replicate in bacteria. The vector is constructed so that (i) the DNA segment is placed adjacent (or operably linked or "downstream" or "under the control of") to the polyhedron gene promoter and (ii) the promoter/monoterpene synthase combination is flanked on both sides by 200-300 base pairs of baculovirus DNA (the flanking sequences).

To produce the monoterpene synthase DNA construct, a cDNA clone encoding the full length (-)-limonene synthase, (-)-pinene synthase and myrcene synthase is obtained using methods such as those described herein. The DNA construct is contacted in a host cell with baculovirus DNA of an appropriate baculovirus (that is, of the same species of baculovirus as the promoter encoded in the construct) under conditions such that recombination is effected. The resulting recombinant baculoviruses encode the full (-)-limonene synthase, (-)-pinene synthase and myrcene synthase. For example, an insect host cell can be cotransfected or transfected separately with the DNA construct and a functional baculovirus. Resulting recombinant baculoviruses can then be isolated and used to infect cells to effect production of the monoterpene synthase. Host insect cells include, for example, Spodoptera frugiperda cells, that are capable of producing a baculovirus- expressed monoterpene synthase. Insect host cells infected with a recombinant baculovirus of the present invention are then cultured under conditions allowing expression of the baculovirus-encoded (-)-limonene synthase, (-)-pinene synthase and myrcene synthase. (-)-limonene synthase, (-)-pinene synthase and myrcene synthase thus produced are then extracted from the cells using methods known in the art.

Other eukaryotic microbes such as yeasts may also be used to practice this invention. The baker's yeast Saccharomyces cerevisiae, is a commonly used yeast, although several other strains are available. The plasmid YRp7 (Stinchcomb et al., Nature 282:39 [1979J; Kingsman et al., Gene 7:141 [1979]; Tschemper et al., Gene 10:157 [1980]) is commonly used as an expression vector in Saccharomyces. This plasmid contains the trpl gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, such as strains ATCC No. 44,076 and PEP4-1 (Jones, Genetics 85:12 [1977]). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Yeast host cells are generally transformed using the polyethylene glycol method, as described by Hinnen (Proc. Natl. Acad. Sci. USA 75: 1929 [1978]). Additional yeast transformation protocols are set forth in Gietz et al., N.A. R. 20(17) 1425(1992); Reeves et al., FEMS 99(2-3): 193-197, (1992). Suitable promoting sequences in yeast vectors include the promoters for

3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073 [1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 [1968]; Holland et al., Biochemistry 17:4900 [1978]), such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 -phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In the construction of suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.

Cell cultures derived from multicellular organisms, such as plants, may be used as hosts to practice this invention. Transgenic plants can be obtained, for example, by transferring plasmids that encode (-)-limonene synthase, (-)-pinene synthase and myrcene synthase and a selectable marker gene, e.g., the kan gene encoding resistance to kanamycin, into Agrobacterium lumifaciens containing a helper Ti plasmid as described in Hoeckema et al., Nature 303:179-181 [1983] and culturing the Agrobacterium cells with leaf slices of the plant to be transformed as described by An et al., Plant Physiology 81:301-305 [1986]. Transformation of cultured plant host cells is normally accomplished through Agrobacterium lumifaciens, as described above. Cultures of mammalian host cells and other host cells that do not have rigid cell membrane barriers are usually transformed using the calcium phosphate method as originally described by Graham and Van der Eb (Virology 52:546 [1978]) and modified as described in sections 16.32-16.37 of Sambrook et al., supra. However, other methods for introducing DNA into cells such as Polybrene (Kawai and Nishizawa, Mol. Cell. Biol. 4: 1172 [1984]), protoplast fusion (Schaffner, Proc. Nail. Acad. Sci. USA 77:2163 [1980]), electroporation (Neumann et al., EMBOJ. 1:841 [1982]), and direct micro injection into nuclei (Capecchi, Cell 22:479 [1980]) may also be used. Additionally, animal transformation strategies are reviewed in Monastersky G.M. and Robl, J.M., Strategies in Transgenic Animal Science, ASM Press, Washington, D.C., 1995. Transformed plant calli may be selected through the selectable marker by growing the cells on a medium containing, e.g., kanamycin, and appropriate amounts of phytohormone such as naphthalene acetic acid and benzyladenine for callus and shoot induction. The plant cells may then be regenerated and the resulting plants transferred to soil using techniques well known to those skilled in the art.

In addition, a gene regulating (-)-limonene synthase, (-)-pinene synthase and myrcene synthase production can be incorporated into the plant along with a necessary promoter which is inducible. In the practice of this embodiment of the invention, a promoter that only responds to a specific external or internal stimulus is fused to the target cDNA. Thus, the gene will not be transcribed except in response to the specific stimulus. As long as the gene is not being transcribed, its gene product is not produced.

An illustrative example of a responsive promoter system that can be used in the practice of this invention is the glutathione-S-transferase (GST) system in maize. GSTs are a family of enzymes that can detoxify a number of hydrophobic electrophilic compounds that often are used as pre-emergent herbicides (Weigand et al., Plant Molecular Biology 7:235-243 [1986]). Studies have shown that the GSTs are directly involved in causing this enhanced herbicide tolerance. This action is primarily mediated through a specific 1.1 kb mRNA transcription product. In short, maize has a naturally occurring quiescent gene already present that can respond to external stimuli and that can be induced to produce a gene product. This gene has previously been identified and cloned. Thus, in one embodiment of this invention, the promoter is removed from the GST responsive gene and attached to a (-)-limonene synthase, (-)-pinene synthase and myrcene synthase gene that previously has had its native promoter removed. This engineered gene is the combination of a promoter that responds to an external chemical stimulus and a gene responsible for successful production of (-)-limonene synthase, (-)-pinene synthase and myrcene synthase. In addition to the methods described above, several methods are known in the art for transferring cloned DNA into a wide variety of plant species, including gymnosperms, angiosperms, monocots and dicots (see, e.g., Glick and

Thompson, eds., Methods in Plant Molecular Biology, CRC Press, Boca Raton, Florida [1993]). Representative examples include electroporation-facilitated DNA uptake by protoplasts (Rhodes et al., Science 240(4849):204-207 [1988]); treatment of protoplasts with polyethylene glycol (Lyznik et al., Plant Molecular

Biology 13:151-161 [1989]); and bombardment of cells with DNA laden microprojectiles (Klein et al., Plant Physiol. 91:440-444 [1989] and Boynton et al., Science 240(4858): 1534- 1538 [1988]). Additionally, plant transformation strategies and techniques are reviewed in Birch, R.G., Ann Rev Plant Phys Plant Mol

Biol 48:297 (1997); Forester et al., Exp. Agric. 33:15-33 (1997). Minor variations make these technologies applicable to a broad range of plant species.

Each of these techniques has advantages and disadvantages. In each of the techniques, DNA from a plasmid is genetically engineered such that it contains not only the gene of interest, but also selectable and screenable marker genes. A selectable marker gene is used to select only those cells that have integrated copies of the plasmid (the construction is such that the gene of interest and the selectable and screenable genes are transferred as a unit). The screenable gene provides another check for the successful culturing of only those cells carrying the genes of interest. A commonly used selectable marker gene is neomycin phosphotransferase II (NPT II). This gene conveys resistance to kanamycin, a compound that can be added directly to the growth media on which the cells grow. Plant cells are normally susceptible to kanamycin and, as a result, die. The presence of the NPT II gene overcomes the effects of the kanamycin and each cell with this gene remains viable. Another selectable marker gene which can be employed in the practice of this invention is the gene which confers resistance to the herbicide glufosinate (Basta). A screenable gene commonly used is the β-glucuronidase gene (GUS). The presence of this gene is characterized using a histochemical reaction in which a sample of putatively transformed cells is treated with a GUS assay solution. After an appropriate incubation, the cells containing the GUS gene turn blue.

The plasmid containing one or more of these genes is introduced into either plant protoplasts or callus cells by any of the previously mentioned techniques. If the marker gene is a selectable gene, only those cells that have incorporated the DNA package survive under selection with the appropriate phytotoxic agent. Once the appropriate cells are identified and propagated, plants are regenerated. Progeny from the transformed plants must be tested to insure that the DNA package has been successfully integrated into the plant genome.

Mammalian host cells may also be used in the practice of the invention. Examples of suitable mammalian cell lines include monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney Hne 293S (Graham et al., J. Gen. Virol. 36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (Urlab and Chasin, Proc. Natl. Acad. Sci USA 77:4216 [1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243 [1980]); monkey kidney cells (CVI-76, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL 51); rat hepatoma cells (HTC, MI.54, Baumann et al., J. Cell Biol 85: 1 [1980]); and TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383:44 [1982]). Expression vectors for these cells ordinarily include (if necessary) DNA sequences for an origin of replication, a promoter located in front of the gene to be expressed, a ribosome binding site, an RNA splice site, a polyadenylation site, and a transcription terminator site.

Promoters used in mammalian expression vectors are often of viral origin. These viral promoters are commonly derived from polyoma virus, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The SV40 virus contains two promoters that are termed the early and late promoters. These promoters are particularly useful because they are both easily obtained from the virus as one DNA fragment that also contains the viral origin of replication (Fiers et al, Nature 273:113 [1978]). Smaller or larger SV40 DNA fragments may also be used, provided they contain the approximately 250-bp sequence extending from the Hindlll site toward the Bgll site located in the viral origin of replication. Alternatively, promoters that are naturally associated with the foreign gene

(homologous promoters) may be used provided that they are compatible with the host cell line selected for transformation.

An origin of replication may be obtained from an exogenous source, such as

SV40 or other virus (e.g., Polyoma, Adeno, VSV, BPV) and inserted into the cloning vector. Alternatively, the origin of replication may be provided by the host cell chromosomal replication mechanism. If the vector containing the foreign gene is integrated into the host cell chromosome, the latter is often sufficient.

The use of a secondary DNA coding sequence can enhance production levels of (-)-limonene synthase, (-)-pinene synthase and myrcene synthase in transformed cell lines. The secondary coding sequence typically comprises the enzyme dihydrofolate reductase (DHFR). The wild-type form of DHFR is normally inhibited by the chemical methotrexate (MTX). The level of DHFR expression in a cell will vary depending on the amount of MTX added to the cultured host cells. An additional feature of DHFR that makes it particularly useful as a secondary sequence is that it can be used as a selection marker to identify transformed cells. Two forms of DHFR are available for use as secondary sequences, wild-type DHFR and MTX- resistant DHFR. The type of DHFR used in a particular host cell depends on whether the host cell is DHFR deficient (such that it either produces very low levels of DHFR cndogcnously, or it does not produce functional DHFR at all). DHFR-deficient cell lines such as the CHO cell line described by Urlaub and Chasin, supra, are transformed with wild-type DHFR coding sequences. After transformation, these DHFR-deficient cell lines express functional DHFR and are capable of growing in a culture medium lacking the nutrients hypoxanthine, glycine and thymidine. Nontransformed cells will not survive in this medium. The MTX-resistant form of DHFR can be used as a means of selecting for transformed host cells in those host cells that endogenously produce normal amounts of functional DHFR that is MTX sensitive. The CHO-K1 cell line (ATCC No. CL 61) possesses these characteristics, and is thus a useful cell line for this purpose. The addition of MTX to the cell culture medium will permit only those cells transformed with the DNA encoding the MTX-resistant DHFR to grow. The nontransformed cells will be unable to survive in this medium.

Prokaryotes may also be used as host cells for the initial cloning steps of this invention. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated. Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli X1776 (ATCC No. 31 ,537), and E. coli B; however many other strains of E. coli, such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts. Prokaryotic host cells or other host cells with rigid cell walls are preferably transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells. Prokaryote transformation techniques are set forth in Dower, W.J., in Genetic Engineering, Principles and Methods, 12:275-296, Plenum Publishing Corp., 1990; Hanahan et al., Meth. Enxymol., 204:63 (1991).

As a representative example, cDNA sequences encoding (-)-limonene synthase, (-)-pinene synthase or myrcene synthase may be transferred to the (His)6*Tag pET vector commercially available (from Novagen) for overexpression in E. coli as heterologous host. This pET expression plasmid has several advantages in high level heterologous expression systems. The desired cDNA insert is ligated in frame to plasmid vector sequences encoding six histidines followed by a highly specific protease recognition site (thrombin) that are joined to the amino terminus codon of the target protein. The histidine "block" of the expressed fusion protein promotes very tight binding to immobilized metal ions and permits rapid purification of the recombinant protein by immobilized metal ion affinity chromatography. The histidine leader sequence is then cleaved at the specific proteolysis site by treatment of the purified protein with thrombin, and the (-)-limonene synthase, (-)-pinene synthase and myrcene synthase again purified by immobilized metal ion affinity chromatography, this time using a shallower imidazole gradient to elute the recombinant synthases while leaving the histidine block still adsorbed. This overexpression-purification system has high capacity, excellent resolving power and is fast, and the chance of a contaminating E. coli protein exhibiting similar binding behavior (before and after thrombin proteolysis) is extremely small.

As will be apparent to those skilled in the art, any plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell may also be used in the practice of the invention. The vector usually has a replication site, marker genes that provide phenotypic selection in transformed cells, one or more promoters, and a polylinker region containing several restriction sites for insertion of foreign DNA. Plasmids typically used for transformation of E. coli include pBR322, pUC18, pUC19, pUCI18, pUC119, and Bluescript M13, all of which are described in sections 1.12-1.20 of Sambrook et al., supra. However, many other suitable vectors are available as well. These vectors contain genes coding for ampicillin and/or tetracycline resistance which enables cells transformed with these vectors to grow in the presence of these antibiotics.

The promoters most commonly used in prokaryotic vectors include the β-lactamase (penicillinase) and lactose promoter systems (Chang et al. Nature 375:615 [1978]; Itakura et al., Science 198:1056 [1977]; Goeddel et al., Nature 281:544 [1979]) and a tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 [1980]; EPO Appl. Publ. No. 36,776), and the alkaline phosphatase systems. While these are the most commonly used, other microbial promoters have been utilized, and details concerning their nucleotide sequences have ■ been published, enabling a skilled worker to ligate them functionally into plasmid vectors (see Siebenlist et al., Cell 20:269 [1980]).

Many eukaryotic proteins normally secreted from the cell contain an endogenous secretion signal sequence as part of the amino acid sequence. Thus, proteins normally found in the cytoplasm can be targeted for secretion by linking a signal sequence to the protein. This is readily accomplished by ligating DNA encoding a signal sequence to the 5' end of the DNA encoding the protein and then expressing this fusion protein in an appropriate host cell. The DNA encoding the signal sequence may be obtained as a restriction fragment from any gene encoding a protein with a signal sequence. Thus, prokaryotic, yeast, and eukaryotic signal sequences may be used herein, depending on the type of host cell utilized to practice the invention. The DNA and amino acid sequence encoding the signal sequence portion of several eukaryotic genes including, for example, human growth hormone, proinsulin, and proalbumin are known (see Stryer, Biochemistry W.H. Freeman and Company, New York, NY, p. 769 [1988]), and can be used as signal sequences in appropriate eukaryotic host cells. Yeast signal sequences, as for example acid phosphatase (Arima et al., Nuc. Acids Res. 11:1657 [1983]), α-factor, alkaline phosphatase and invertase may be used to direct secretion from yeast host cells. Prokaryotic signal sequences from genes encoding, for example, LamB or O pF (Wong et al., Gene 68:193 [1988]), MalE, PhoA, or beta-lactamase, as well as other genes, may be used to target proteins from prokaryotic cells into the culture medium. Trafficking sequences from plants, animals and microbes can be employed in the practice of the invention to direct the monoterpene synthase proteins of the present invention to the cytoplasm, endoplasmic reticulum, mitochondria or other cellular components, or to target the protein for export to the medium. These considerations apply to the overexpression of (-)-limonene synthase, (-)-pinene synthase and myrcene synthase, and to direction of expression within cells or intact organisms to permit gene product function in any desired location.

The construction of suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes and the monoterpene synthase DNA of interest are prepared using standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well known in the art (see, for example, Maniatis, supra, and Sambrook et al., supra).

As discussed above, (-)-limonene synthase, (-)-pinene synthase and myrcene synthase variants are preferably produced by means of mutation(s) that are generated using the method of site-specific mutagenesis. This method requires the synthesis and use of specific oligonucleotides that encode both the sequence of the desired mutation and a sufficient number of adjacent nucleotides to allow the oligonucleotide to stably hybridize to the DNA template. The foregoing may be more fully understood in connection with the following representative examples, in which "Plasmids" are designated by a lower case p followed by an alphanumeric designation. The starting plasmids used in this invention are either commercially available, publicly available on an unrestricted basis, or can be constructed from such available plasmids using published procedures. In addition, other equivalent plasmids are known in the art and will be apparent to the ordinary artisan.

"Digestion", "cutting" or "cleaving" of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at particular locations in the DNA. These enzymes are called restriction endonucleases, and the site along the DNA sequence where each enzyme cleaves is called a restriction site. The restriction enzymes used in this invention are commercially available and are used according to the instructions supplied by the manufacturers. (See also sections 1.60-1.61 and sections 3.38-3.39 of Sambrook et al, supra.)

"Recovery" or "isolation" of a given fragment of DNA from a restriction digest means separation of the resulting DNA fragment on a polyacrylamide or an agarose gel by electrophoresis, identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the gel from DNA. This procedure is known generally. For example, see Lawn et al. (Nucleic Acids Res. 9:6103-6114 [1982]), and Goeddel et al. (Nucleic Acids Res., supra).

The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention. All literature citations herein are expressly incorporated by reference.

EXAMPLE 1 PCR-Based Generation of Probes for Cloning Monoterpene Synthases From

Grand fir (Abies grandis) Substrates, Reagents and cDNA Library - [1- H]Geranyl diphosphate (250 Ci/mol) (Croteau, R., Alonso, W.R., Koepp, A.E., and Johnson, M.A. (1994) Arch. Biochem. Biophys. 309:184-192), [l-³H]farnesyl diphosphate (125 Ci/mol) (Dehal, S.S., and Croteau, R. (1988) Arch. Biochem. Biophys. 261:346-356) and [l-^' l ljgeranylgeranyl diphosphate (120 Ci/mol) (LaFever, R.E., Stofer Vogel, B., and Croteau, R. (1994) Arch. Biochem. Biophys. 313:139-149) were prepared as described previously. Terpenoid standards were from our own collection. All other biochemicals and reagents were purchased from Sigma Chemical Co. or Aldrich Chemical Co., unless otherwise noted. Construction of the λZAP II cDNA library, using mRNA isolated from wounded Grand fir sapling stems, was described previously (Stofer Vogel, B., Wildung, M.R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268).

PCR-Based Probe Generation - Based on comparison of sequences of limonene synthase from spearmint (Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J., and Croteau, R. (1993) J. Biol. Chem. 268:23016- 23024), 5-ty;/-aristolochene synthase from tobacco (Facchini, P.J., and Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092), and casbene synthase from castor bean (Mau, C.J.D., and West, CA. (1994) Proc. Natl. Acad. Sci. USA 91:8497-8501), four conserved regions were identified for which a set of consensus degenerate primers were synthesized: Primer A (SEQ ID NO:7); Primer B (SEQ ID NO:8); Primer C (SEQ ID NO:9); Primer D (SEQ ID NO: 10). Primers A (SEQ ID NO:7), B (SEQ ID NO:8) and C (SEQ ID NO:9) have been described previously (Steele, C, Levvinsohn, E. and Croteau, II,, Proc. Nat'l. Acad. Sci. USA, 92: 4164-4168 (1995)); primer D (SEQ ID NO: 10) was designed based on the conserved amino acid sequence motif DD(T/I)(I/Y/F)D(A/V)Y(A/G)(SEQ ID NO:25) of the above noted terpene synthases (Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024; Facchini, P.J., and Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092; Mau, C.J.D., and West, CA. (1994) Proc. Natl. Acad. Sci. USA 91:8497-8501).

Each of the sense primers, A (SEQ ID NO:7), B (SEQ ID NO:8) and D (SEQ ID NO: 10), was used for PCR in combination with antisense primer C (SEQ ID NO:9) by employing a broad range of amplification conditions. PCR was performed in a total volume of 50 μl containing 20 mM Tris/HCl (tris(hydroxymethyl) aminomethane/HCl, pH 8.4), 50 mM KC1, 5 mM MgCl₂, 200 μM of each dNTP, 1-5 μM of each primer, 2.5 units of Taq polymerase (BRL) and 5 μl of purified Grand fir

9 stem cDNA library phage as template (1.5 x 10 pfu ml). Analysis of the PCR reaction products by agarose gel electrophoresis (Sambrock, J., Fritsch, E.F., and Maniatis, T. (1 89) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) revealed that only the combination of primers C and D generated a specific PCR product of approximately 110 bps (base pairs). This PCR product was gel purified, ligated into pT7Blue (Novagen), and transformed into E. coli XL 1 -Blue cells. Plasmid DNA was prepared from 41 individual transformants and the inserts were sequenced (DyeDeoxy Terminator Cycle Sequencing, Applied Biosystems). Four different insert sequences were identified, and were designated as probes 1 (SEQ ID NO:l 1), 2 (SEQ ID NO: 12), 4 (SEQ ID NO: 13) and 5 (SEQ ID NO: 14). Subsequent isolation of four new cDNA species (AG1.28 (SEQ ID NO: 15);

AG2.2 (SEQ ID NO:l); AG4.30 (SEQ ID NO:17) and AG5.9 (SEQ ID NO:19)), encoding terpene synthases from Grand fir corresponding to probes 1 (SEQ ID NO: l l), 2 (SEQ ID NO: 12), 4 (SEQ ID NO:13) and 5 (SEQ ID NO: 14), respectively, allowed the identification of three additional conserved sequence elements which were used to design a set of three new PCR primers: Primer E (5'-GGI GA(A/G) A(A/C)(A/G) (A/G)TI ATG GA(A/G) GA(A/G) GC-3')(SEQ ID NO:21); Primer F (5'-GA(A/G) (C/T)TI CA(G/A) (C/T)TI (A/C/T)(C/G/T)I (A/C)GI TGG TGG-3'XSEQ ID NO:22) and Primer G (5'-CCA (A/G)TT IA(A/G) ICC (C/T)TT IΛC (A/G)TC-3')(SEQ ID NO:23). Degenerate primer E (SEQ ID NO:21) was designed to conserved element

GE(K/T)(V/I)M(E/D)EA (SEQ ID NO:26) and degenerate primer F (SEQ ID NO:22) was designed to conserved element Q(F/Y/D)(I/L)(T/L/R)RWW (SEQ ID NO:27) by comparing the sequences of five cloned terpene synthases from Grand fir: a monoterpene synthase corresponding to probe 2 (SEQ ID NO: 12), two sesquiterpene synthases corresponding to probe 4 (SEQ ID NO:13) and probe 5 (SEQ ID NO:14), respectively, a previously described diterpene synthase (Stofer Vogel, B., Wildung,

M.R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268), and a truncated terpene synthase corresponding to probe 1 (SEQ ID NO: 11). Degenerate primer G (SEQ ID NO:23) was designed according to the amino acid sequence DVIKG(F/L)NW (SEQ ID NO:28) obtained from a peptide generated by trypsin digestion of purified (-)-pinene synthase from Grand fir. Primers E (SEQ ID NO:21) and F (SEQ ID NO:22) were independently used for PCR amplification in combination with primer G (SEQ ID NO:23), with Grand fir stem cDNA library as template. The combination of primers E (SEQ ID NO:21) and G (SEQ ID NO:23) yielded a specific PCR product of approximately 1020 bps. This PCR product was ligated into pT7Blue and transformed into E. coli XL 1 -Blue. Plasmid DNA was prepared from 20 individual transformants and inserts were sequenced from both ends. The sequence of this 1022 bp insert was identical for all 20 plasmids and was designated as probe 3 (SEQ ID NO:24). EXAMPLE 2

Screening a Wounded Grand fir Stem cDNA Library For library screening, 100 ng of each probe was amplified by PCR, gel

32 purified, randomly labeled with [α- P]dATP (Feinberg, A.P., and Vogelstein, B. (1984) Anal. Biochem. 137:266-267), and used individually to screen replica filters of 10 plaques of the wound-induced Grand fir stem cDNA library plated on E. coli LE392. Hybridization with probes 1 (SEQ ID NO:l l), 2 (SEQ ID NO:12), 4 (SEQ ID NO:13) and 5 (SEQ ID NO:14) was performed for 14 h at 65 °C in 3 x SSPE and 0.1% SDS. Filters were washed three times for 10 min at 55 °C in 3 x SSPE with 0.1% SDS and exposed for 12 h to Kodak XAR film at -70 °C All of the λZAPII clones yielding positive signals were purified through a second round of hybridization (probe 1 (SEQ ID NO:l 1) gave 25 positives, probe 2 (SEQ ID NO: 12) gave 16 positives, probe 4 (SEQ ID NO: 13) gave 49 positives and probe 5 (SEQ ID NO: 14) gave 12 positives).

Hybridization with probe 3 (SEQ ID NO:24) was performed as before, but the filters were washed three times for 10 min at 65 °C in 3 x SSPE and 0.1% SDS before exposure. Approximately 400 λZAPII clones yielded strong positive signals, and 34 of these were purified through a second round of hybridization at 65°C Approximately 400 additional clones yielded weak positive signals with probe 3 (SEQ ID NO:24), and 18 of these were purified through a second round of hybridization for 20 h at 45 °C Purified λZAP II clones isolated using all five probes were in vivo excised as Bluescript II SK(-) phagemids and transformed into E. coli XLOLR according to the manufacturer's instructions (Stratagene). The size of each cDNA insert was determined by PCR using T3 (SEQ ID NO:29) and T7 (SEQ ID NO:30) promoter primers and selected inserts (>1.5 kb) were partially sequenced from both ends.

EXAMPLE 3 Grand fir Monoterpene Synthase cDNA Expression in E. coli and Enzyme

Assays Except for cDNA clones AG3.18 (SEQ ID NO:3) and AG3.48 (SEQ ID NO:31), all of the partially sequenced inserts were either truncated at the 5'-end, or were out of frame, or bore premature stop codons upstream of the presumptive methionine start codon. For the purpose of functional expression, a 2023 bp insert fragment, extending from nucleotides 75 to 2097 of the sequence set forth in SEQ ID NO: l , and a 1911 bp insert fragment, extending from nucleotide 1 to nucleotide 1910 of the sequence set forth in SEQ ID NO:3, were subcloned in frame into pGEX vectors (Pharmacia). A 2016 bp fragment extending from nucleotide 73 to nucleotide 2088 of the sequence set forth in SEQ ID NO:5 was subcloned in frame into the pSBETa vector (Schenk, P.M., Baumann, S., Mattes, R., and Steinbiss, H.-H. (1995) Biotechniques 19, 196-200). To introduce suitable restriction sites for subcloning, fragments were amplified by PCR using primer combinations 2.2-BamUl (5^*-CAA AGG GAT CCA GAA TGG CTC TGG-3')(SEQ ID NO:33) and 2.2-Notl (5'-AGT AAG CGG CCG CTT TTT AAT CAT ACC CAC-3')(SEQ ID NO:34) with pAG2.2 insert (SEQ ID NO: l) as template, 3.18-EcoRl (5^*-CTG CAG GAA TTC GGC ACG AGC-3')(SEQ ID NO:35) and 3A8-Smal (5'-CAT AGC CCC GGG CAT AGA TTT GAG CTG-3')(SEQ ID NO:36) with pAG3.18 insert (SEQ ID NO:3) as template, and 10-Ndel (5-GGC AGG AAC ATA TGG CTC TCC TTT CTA TCG- 3')(SEQ ID NO:37) and 10-BamUl (5'-TCT AGA ACT AGT GGATCC CCC GGG CTG CAG-3')(SEQ ID NO:38) with pAGlO insert (SEQ ID NO:5) as template.

PCR reactions were performed in volumes of 50 μl containing 20 mM Tiϊs/I ICl (pi I 8.8), 10 mM KC1, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1% Triton X-100, 5 μg bovine serum albumin (BSA), 200 μM of each dNTP, 0.1 μM of each primer, 2.5 units of recombinant Pfu polymerase (Stratagene) and 100 ng plasmid DNA with the following program: denaturation at 94 °C, 1 min; annealing at 60 °C, 1 min; extension at 72 °C, 3.5 min; 35 cycles with final extension at 72 °C, 5 min. The PCR products were purified by agarose gel electrophoresis and used as template for a secondary PCR amplification with the identical conditions in total volumes of 250 μl each. Products from this secondary amplification were digested with the above indicated restriction enzymes, purified by ultrafiltration and then ligated, respectively, into ifømHI/ΛOtl-digested pGEX-4T-2 to yield plasmid pGAG2.2, into EcoRl/Sma -digested pGEX-4T-3 to yield plasmid pGAG3.18, and into /VJel βαmHI-digested pSBETa to yield plasmid pSBAGlO; these plasmids were then transformed into E. coli XL 1 -Blue or E. coli BL21(DE3).

For expression, bacterial strains E. coli XLOLR pAG3.18, E. coli

XLOLR/pAG3.48, E. coli XLl-Blue/pGAG2.2, E. coli XLl-Blue/pGAG3.18, and E. coli BL21(DE3)/pSBAG10 were grown to A_6ϋ0 = 0.5 at 37 °C in 5 ml of LB medium (Sambrock, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A

Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,

NY) supplemented with 100 μg ampicillin ml or 30 μg kanamycin/ml as determined by the vector. Cultures were then induced by addition of 1 mM isopropyl-1-thio-β-D-galactopyranoside and grown for another 12 h at 20 °C Cells were harvested by centrifugation (2000 x g, 10 min) and resuspended in either 1 ml monoterpene synthase assay buffer [50 mM Tris/HCl (pH 7.5), 500 mM KC1, 1 mM

MnCl₂, 5 mM dithiothreitol, 0.05% (w/v) NaHSO₃ and 10% (v/v) glycerol], 1 ml sesquiterpene synthase assay buffer [10 mM dibasic potassium phosphate, 1.8 mM monobasic potassium phosphate (pH 7.3), 140 mM NaCl, 10 mM MgCl₂, 5 mM dithiothreitol, 0.05% (w/v) NaHSO₃ and 10% (v/v) glycerol], or 1 ml diterpene synthase assay buffer [30 mM Hepes (jV-2-hydroxyethylpiperazine-.Λ/'-2- elhanesulfonic acid, pH 7.2), 7.5 mM MgCl₂, 5 mM dithiothreitol, 10 μM MnCl₂,

0.05% (w/v) NaHSO₃ and 10% (v/v) glycerol]. Cells were disrupted by sonication (Braun-Sonic 2000 with microprobe at maximum power for 15 seconds at 0-4°C), the homogenates were cleared by centrifugation (18,000 x g, 10 min), and 1 ml of the resulting supernatant was assayed for monoterpene synthase activity with 2.5 μM of [1- H] geranyl

₃ diphosphate, for sesquiterpene synthase activity with 3.5 μM [1- H]farnesyl

₃ diphosphate, or for diteφene synthase activity with 5 μM [1- H]geranylgeranyl diphosphate following standard protocols (Croteau, R., and Cane, D.E. (1985)

Methods Enzymol 110:383-405; LaFever, R.E., Stofer Vogel, B., and Croteau, R.

(1994) Arch. Biochem. Biophys. 313:139-149; Dehal, S.S., and Croteau, R. (1988)

Arch. Biochem. Biophys. 261:346-356). In the case of the monoterpene synthase and sesquiterpene synthase assays, the incubation mixture was overlaid with 1 ml pentane to trap volatile products. In all cases, after incubation at 31°C for 2 h, the reaction mixture was extracted with pentane (3 x 1 ml) and the combined extract was passed through a 1.5 ml column of anhydrous MgSO and silica gel (Mallinckrodt 60 A) to provide the terpene hydrocarbon fraction free of oxygenated metabolites. The columns were subsequently eluted with 3 x 1 ml of ether to collect any oxygenated products, and an aliquot of each fraction was taken for liquid scintillation counting to determine conversion rate.

Product Identification - To obtain sufficient product for analysis by radio- GLC (gas liquid chromatography), chiral capillary GLC and GLC-MS (mass spectrum spectrometry), preparative-scale enzyme incubations were carried out. Thus, the enzyme was prepared from 50 ml of cultured bacterial cells by extraction with 3 ml of assay buffer as above, and the extracts were incubated with excess substrate overnight at 31°C The hydrocarbon fraction was isolated by elution through MgSO -silica gel as before, and the pentane eluate was concentrated for evaluation by capillary radio-GLC as described (Croteau, R., and Satterwhite, D.M. (1990) J. Chromalogr. 500:349-354), by chiral column capillary GLC (Lewinsohn, E., Savage, T.J., Gijzen, M., and Croteau, R. (1993) Phylochem. Anal. 4:220-225), and by combined GLC-MS [Hewlett-Packard 6890 GC-MSD with cool (40°C) on- column injection, detection via electron impact ionization (70 eV), He carrier at 0.7 psi., column: 0.25 mm i.d. x 30 m fused silica with 0.25 μm film of 5MS (Hewlett- Packard) programmed from 35°C (5 min hold) to 230°C at 5°C/min].

EXAMPLE 4 Sequence Analysis Inserts of all recombinant bluescript plasmids and pGEX plasmids were completely sequenced on both strands via primer walking and nested deletions (Sambrock, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) using the DyeDeoxy Terminator Cycle Sequencing method (Applied Biosystems). Sequence analysis was done using the Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, WI.

EXAMPLE 5 RNA Extraction and Northern Blotting Grand fir sapling stem tissue was harvested prior to wounding or two days after wounding by a standard procedure (Gijzen, M., Lewinsohn, E., and Croteau, R. ( 1991 ) Arch. Biochem. Biophys. 289:267-273). Total RNA was isolated (Lewinsohn, E., Steele, C.L., and Croteau, R. (1994) Plant Mol. Biol. Rep. 12:20-25) and 20 μg of RNA per gel lane was separated under denaturing conditions (Sambrock, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and transferred to nitrocellulose membranes (Schleicher and Schuell) according to the manufacturer's protocol. To prepare hybridization probes, cDNA fragments of 1.4-1.5 kb were amplified by PCR from AG2.2 (SEQ ID NO: 1) with primer JB29 (5'-CTA CCA TTC CAA TAT CTG-3')(SEQ ID NO:39) and primer 2-8 (5'-GTT GGA TCT TAG AAG TTC CC-3')(SEQ ID NO:40), from AG3.18 (SEQ ID NO:3) with primer 3-9 (5'-TTT CCA TTC CAA CCT CTG GG-3')(SEQ ID NO:41) and primer 3-11 (5'-CGT AAT GGA AAG CTC TGG CG-3')(SEQ ID NO:42), and from AGIO (SEQ ID NO:5) with primer 7-1 (5'-CCT TAC ACG CCT TTG GAT GG-3')(SEQ ID NO:43) and primer 7-3 (5'-TCT GTT GAT CCA GGA TGG TC-3')(SEQ ID NO:44). The probes were randomly labeled with [α- 32 P]dATP (Feinberg, A.P., and Vogelstein, B. (1984) Anal. Biochem. 137:266-267). Blots were hybridized for 24 h at 55°C in 3 x SSPE and 0.1% SDS, washed at 55°C in 1 x SSPE and 0.1%, SDS and subjected to autoradiography as described above at -80°C for 24 h.

EXAMPLE 6 Cloning and Characterization of Clones AG1.28 (SEQ ID NO:15), AG2.2 (SEQ ID NO:l), AG4.30 (SEQ ID NO:17) and AG 5.9 tSEO ID NO:19)

Similarity-Based Cloning of Grand fir Terpene Synthases - Grand fir has been developed as a model system for the study of induced oleoresin production in conifers in response to wounding and insect attack (Johnson, M.A., and Croteau, R. (1987) in Ecology and Metabolism of Plant Lipids (Fuller, G., and Nes, W.D., eds) pp. 76-91, American Chemical Society Symposium Series 325, Washington, DC; Gijzen, M., Lewinsohn, E., Savage, T.J., and Croteau, R.B. (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., Buttery, R.G., and Sugisawa, H., eds) pp. 8-22, American Chemical Society Symposium Series 525, Washington, DC; Raffa, K. F., and Berryman, A.A. (1982) Can. Entomol. 114:797-810; Steele, C, Lewinsohn, E., and Croteau, R. (1995) Proc. Nail Acad. Sci. USA 92:4164- 4168; Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) in Regulation of lsopenlenoid Metabolism (Nes, W.D., Parish, E.J., and Trzaskos, J.M., eds) pp. 8-17, American Chemical Society Symposium Series 497, Washington, DC). The chemistry and biosynthesis of the oleoresin monoterpenes, sesquiterpenes and diterpenes have been well defined (Lewinsohn, E., Savage, T.J., Gijzen, M., and Croteau, R. (1993) Phytochem. Anal. 4:220-225; Lewinsohn, E., Gijzen, M., and Croteau, R. (1991) Plant Physiol 96:44-49; Funk, C, Lewinsohn, E., Stofer Vogel, B., Steele C, and Croteau, R. (1994) Plant Physiol. 106:999-1005; Gijzen, M., Lewinsohn, I-., and Crolemi, R. (1991) Arch. Biochem. Biophys. 289:267-273; Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173; LaFever, R.E., Stofer Vogel, B., and Croteau, R. (1994) Arch. Biochem. Biophys. 313:139-149; Funk, C, and Croteau, R. (1994) Arch. Biochem. Biophys. 308:258-266); however, structural analysis of the responsible terpene synthases as well as studies on the regulation of oleoresinosis require the isolation of cDN A species encoding the terpene synthases. Protein purification from conifers, as the basis for cDNA isolation, has been of limited success (Stofer Vogel, B., Wildung, M.R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268) and thus far has not permitted cloning of any of the monoterpene synthases from these species (Steele, C, Lewinsohn, E., and Croteau, R. (1995) Proc. Natl. Acad. Sci. (7&4 92:4164-4168).

As a possible alternative to protein-based cloning of terpene synthases, a homology-based PCR strategy was proposed (Steele, C, Lewinsohn, E., and Croteau, R. (1995) Proc. Natl. Acad. Sci. USA 92:4164-4168) that was founded upon the three terpene synthases of plant origin then available, a monoterpene synthase, (-)-45'-limonene synthase, from spearmint (Mentha spicata, Lamiaceae) (Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024), a sesquiterpene synthase, 5-eτ?/^'-aristolochene synthase, from tobacco (Nicotiana labacum, Solanaceae) (Facchini, P.J., and Chappell, J. (1992) Proc. Nail Acad. Sci. USA 89:11088-11092), and a diterpene synthase, casbene synthase, from castor bean (Ricinus communis, Euphorbiaceae) (Mau, C.J.D., and West, CA. (1994) Proc. Natl. Acad. Sci. USA 91:8497-8501). Despite the taxonomic distances between these three angiosperm species and the differences in substrate utilized, reaction mechanism and product type of the three enzymes, a comparison of the deduced amino acid sequences identified several conserved regions that appeared to be useful for the design of degenerate PCR primers (see Example 1). Using cDNA from a wound-induced Grand fir stem library as template, PCR primers C (SEQ ID NO:9) and D (SEQ ID NO: 10) amplified products corresponding to four distinct sequence groups, all of which showed significant similarity to sequences of cloned terpene synthases of plant origin. The four different inserts were designated as probes 1 (SEQ ID NO: 1 1), 2 (SEQ ID NO: 12), 4 9/02030

(SEQ ID NO:13) and 5 (SEQ ID NO:14), and were employed for isolation of the corresponding cDNA clones by plaque hybridization.

Screening of 10 cDNA phage plaques from the wounded Grand fir stem library, with each of the four probes, yielded a four-fold difference in the number of positives, most likely reflecting different levels of expression of the corresponding genes. Size selected inserts (>1.5 kb) of purified and in vivo excised clones were partially sequenced from both ends, and were shown to segregate into four distinct groups corresponding to the four hybridization probes. Since all cDNAs corresponding to probes 1 (SEQ ID NO: 11), 4 (SEQ ID NO: 13) and 5 (SEQ ID NO: 14) were truncated at their 5'-ends, only inserts of the largest representatives of each group, clone AG1.28 (SEQ ID NO:15), clone AG2.2 (SEQ ID NO:l) (apparently full length), clone AG4.30 (SEQ ID NO:17) and clone AG5.9 (SEQ ID NO: 19), were completely sequenced. Clone AG1.28 (SEQ ID NO:15)(2424 bps) includes an open reading frame (ORF) of 2350 nucleotides (nts) encoding 782 amino acids (SEQ ID NO: 16); clone AG2.2 (SEQ ID NO:l)(2196 bps), includes an ORF of 1881 nts encoding 627 amino acids (SEQ ID NO:2); clone AG4.30 (SEQ ID NO: 17)(1967 bps) includes an ORF of 1731 nts encoding 577 amino acids (SEQ ID NO: 18) and clone AG5.9 (SEQ ID NO:19)(1416 bps) includes an ORF of 1194 nucleotides encoding 398 amino acids (SEQ ID NO:20). cDNA clones AG1.28 (SEQ ID NO: 15), AG2.2 (SEQ ID NO:l), AG4.30

(SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19) were compared pairwise with each other and with other cloned plant terpene synthases. Truncated clone AG1.28 (SEQ ID NO: 15) resembled most closely in size and sequence (72% similarity, 49% identity) a diterpene cyclase, abietadiene synthase, from Grand fir (Stofer Vogel, B., Wildung, M.R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268). Clones AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19) share approximately 80% similarity (60% identity) at the amino acid level, and are almost equally distant from both clone AG1.28 (SEQ ID NO: 15) and full-length clone AG2.2 (SEQ ID NO:l)(range of 65-70% similarity and 45-47% identity); the amino acid sequence similarity between AG1.28 (SEQ ID NO: 15) and AG2.2 (SEQ ID NO:l) is 65% (41% identity). Considering the high level of homology between AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19), these comparisons suggest that the four new cDNAs, AG1.28 (SEQ ID NO:15), AG2.2 (SEQ ID NOT), AG4.30 (SEQ ID NO: 17) and AG5.9 (SEQ ID NO: 19), represent the three major subfamilies of Grand fir teφene synthase genes encoding monoterpene synthases, sesquiterpene synthases and diterpene synthases.

Identification ofcDNA Clone AG2.2 (SEQ ID NOT) as Myrcene Synthase - The pAG2.2 insert (SEQ ID NOT) appeared to be a full-length clone encoding a protein of molecular weight 72,478 with a calculated pi at 6.5. The size of the translated protein encoded by AG2.2 (SEQ ID NOT) (627 residues)(SEQ ID NO:2) is in the range of the monoterpene synthase preproteins for limonene synthase from spearmint (Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024) and Perillafrutescens (Yuba, A., Yazaki, K., Tabata, M., Honda, G., and Croteau, R. (1996) Arch. Biochem. Biophys. 332:280- 287), but is about 240 amino acids shorter than the two gymnosperm diterpene synthase preproteins for abietadiene synthase (Stofer Vogel, B., Wildung, M.R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268) and taxadiene synthase (Wildung, M.R., and Croteau, R. (1996) J. Biol. Chem. 271:9201-9204). Monoterpene and diteφene biosynthesis are compartmentalized in plastids whereas sesquiterpene biosynthesis is cytosolic (reviewed in Kleinig, H. (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:39-53; Gershenzon, J., and Croteau, R. (1993) in Lipid Metabolism in Plants (Moore, T.S. Jr., ed) pp. 339-388, CRC Press, Boca Raton, FL; McGarvey, D.J., and Croteau, R. (1995) Plant Cell 7, 1015-1026); thus, monoterpene and diterpene synthases are encoded as preproteins bearing an amino- terminal transit peptide for import of these nuclear gene products into plastids where they are proteolytically processed to the mature forms (Keegstra, K., Olsen, J.J., and Theg, S.M. (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol 40:471-501; von Heijne, G., Stepphuhn, J., and Herrmann (1989) Eur. J. Biochem. 180:535-545). Both the size of the deduced protein and the presence of an N-terminal domain (of 60 to 70 amino acids) with features characteristic of a targeting sequence [rich in serine residues (16-18%) and low in acidic residues (four Asp or Glu) (Keegstra, K., Olsen, J.J., and Theg, S.M. (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:471-501 ; von Heijne, G., Stepphuhn, J., and Herrmann (1989) Eur. J. Biochem. 180:535-545)] suggest that AG2.2 (SEQ ID ΝO:l) encodes a monoterpene synthase rather than a sesquiterpene synthase or a diterpene synthase.

Since pAG2.2 contained the terpene synthase insert in reversed orientation, the ORF was subcloned in frame with glutathione S-transferase, for ultimate ease of purification (Bohlmann, J., DeLuca, V., Eilert, U., and Martin, W. (1995) Plant J. 7:491-501; Bohlmann, J., Lins, T., Martin, W., and Eilert, U. (1996) Plant Physiol 111:507-514), into pGEX-4T-2, yielding plasmid pGAG2.2. The recombinant fusion protein was expressed in E. coli strain XLl-Blue/pGAG2.2, then extracted and assayed for monoterpene synthase, sesquiterpene synthase and diterpene synthase activity using tritium labeled geranyl diphosphate, farnesyl diphosphate and geranylgeranyl diphosphate as the respective substrate. Enzymatic production of a terpene olefin was observed only with geranyl diphosphate as substrate, and the only product was shown to be myrcene by radio-GLC and GLC-MS comparison to an authentic standard (FIGURE 3). Bacteria transformed with pGEX vector containing the AG2.2 insert (SEQ ID NOT) in antisense orientation did not afford detectable myrcene synthase activity when induced, and the protein isolated and assayed as above. A myrcene synthase cDNA has not been obtained previously from any source, although myrcene is a minor co-product (2%) of the native and recombinant limonene synthase from spearmint (Rajaonarivony, J.I.M., Gershenzon, J., and Croteau, R. (1992) Arch. Biochem. Biophys. 296:49-57; Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J., and Croteau, R. (1993) J. Biol. Chem. 268:23016- 23024) and of several enzymes from sage (Croteau, R., and Satterwhite, D.M. (1989) J. Biol. Chem. 264:15309-15315). cDNA cloning and functional expression of myrcene synthase, which is one of several wound-inducible monoterpene synthase activities of Grand fir (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289:267-273), demonstrates that this acyclic monoterpene is formed by a distinct enzyme and is not a co-product of another synthase.

EXAMPLE 7 Cloning and Characterization of Clones AG3.18 (SEQ ID NO:3) Encoding (-)-Pinene Synthase and cDNA Clone AGIO (SEQ ID NO:5) Encoding (-)-Limonene Synthase

Identification of cDNA Clone AG3.18 (SEQ ID NO:3) as (-)-Pinene

Synthase and cDNA Clone AGIO (SEQ ID NO:5) as (-)-Limonene Synthase -

Alignment of the four new terpene synthase cDNA sequences (AG1.28 (SEQ ID

NO: 15), AG2.2 (SEQ ID NOT), AG4.30 (SEQ ID NO:17) and AG.5.9 (SEQ ID NO: 19)), and that for abietadiene synthase (Stofer Vogel, B., Wildung, M.R., Vogel,

G., and Croteau, R. (1996) J. Biol Chem. 271:23262-23268), allowed the identification of several conserved sequence motifs among this enzyme family from

Grand fir, which provided the foundation for an extended similarity-based cloning approach. Two new sense primers E (SEQ ID NO:21) and F (SEQ ID NO:22) were designed according to conserved sequence elements, whereas a degenerate antisense 9/02030

primer G (SEQ ID NO:23) was designed based upon very limited amino acid sequence information from pinene synthase (see Example 1). Only the combination of primers E (SEQ ID NO:21) and G (SEQ ID NO:23) amplified a specific product of 533 bps, which was designated as probe 3 (SEQ ID NO:24). Hybridization of 10⁵ Grand fir λZAP II cDNA clones with probe 3 (SEQ ID

NO:24) yielded two types of signals comprised of about 400 strongly positive clones and an equal number of weak positives, indicating that the probe recognized more than one type of cDNA. Thirty-four of the former clones and 18 of the latter were purified, the inserts were selected by size (2.0-2.5 kb), and the in vivo excised clones were partially sequenced from both ends. Those clones which afforded weak hybridization signals were shown to contain inserts that were either identical to myrcene synthase clone AG2.2 (SEQ ID NOT) or exhibited no significant sequence similarity to terpene synthases. Clone AG3.48 (SEQ ID NO:31) contained the myrcene synthase ORF in the correct orientation and in frame for expression from the Bluescript plasmid vector. This cDNA was functionally expressed in E. coli and the resulting enzyme was shown to accept only geranyl diphosphate as the prenyl diphosphate substrate and to produce myrcene as the exclusive reaction product. This finding with AG3.48 (SEQ ID NO:31) confirms that expression of AG2.2 (SEQ ID NOT) as the glutathione S-transferase fusion protein from pGAG2.2 does not influence substrate utilization or product outcome of the myrcene synthase.

Clones which gave strong hybridization signals segregated into distinct sequence groups represented by clone AG3.18 (SEQ ID NO:3)(2018 bp insert with

ORF of 1884 nt; encoded protein of 628 residues at 71,505 Da and pi of 5.5) and

AGIO (SEQ ID NO:5)(2089 bp insert with ORF of 1911 nt; encoded protein of 637 residues at 73,477 Da and pi of 6.4). AG3.18 (SEQ ID NO:3) and AGIO (SEQ ID

NO:5) form a subfamily together with the myrcene synthase clone AG2.2 (SEQ ID

NOT) that is characterized by a minimum of 79% pairwise similarity (64% identity) at the amino acid level. Like myrcene synthase, both AG3.18 (SEQ ID NO:3) and

AGIO (SEQ ID NO:5) encode N-terminal sequences of 60 to 70 amino acids which are rich in serine (19-22%) and 11-15%), respectively) and low in acidic residues (4 and 2, respectively) characteristic of plastid transit peptides (Keegstra, K., Olsen, J.J., and Theg, S.M. (1989) Annu. Rev. Plant Physiol Plant Mol. Biol. 40:471-501; von Heijne, G., Stepphuhn, J., and Herrmann (1989) Eur. J. Biochem. 180:535-545).

Plasmid pAG3.18 (SEQ ID ΝO:3) contained the presumptive terpene synthase ORF in frame for direct expression from the bluescript plasmid, whereas the 9/02030

AGIO (SEQ ID NO:5) ORF was in reversed orientation. Both AG3.18 (SEQ ID NO:3) and AGIO (SEQ ID NO:5) were subcloned into expression vectors yielding plasmids pGAG3.18 and pSBAGlO. Recombinant proteins were expressed in bacterial strain E. coli XLOLR/pAG3.18, E. coli XLl-Blue/pGAG3T8 and E. coli BL21(DE3)/pSBAG10. When extracts of the induced cells were tested for teφene synthase activity with all of the potential prenyl diphosphate substrates, only geranyl diphosphate was utilized. Extracts from E. coli BL21(DE3)/pSBAG10 converted geranyl diphosphate to limonene as the major product with lesser amounts of α-pinene, β-pinene and β-phellandrene, as determined by radio-GLC and combined GLC-MS (FIGURE 5). Chiral phase capillary GLC on β-cyclodextrin revealed the limonene product to be the (-)-45'-enantiomer and the pinene products to be the related (-)-(15":55 -enantiomers. Although optically pure standards were not available for the analysis, stereochemical considerations suggest that the minor product β-phellandrene is also the mechanistically related (-)-(45)-antipode (Gambliel, II., and Croteau, R. (1984) J. Biol. Chem. 259:740-748; Croteau, R., Satterwhite, D.M., Cane, D.E., and Chang, C.C (1988) J Biol. Chem. 263:10063- 10071 ; Wagschal, K., Savage, T.J., and Croteau, R. (1991) Tetrahedron 47:5933- 5944; Croteau, R., Satterwhite, D.M., Wheeler, C.J., and Felton, N.M. (1989) J. Biol. Chem. 264:2075-2080; LaFever, R.E., and Croteau, R. (1993) Arch. Biochem. Biophys. 301:361_^366). Similar analysis of the monoterpene products generated from geranyl diphosphate by cell-free extracts of E. coli XLOLR/pAG3.18 and E. coli XLl-Blue/pGAG3.18 demonstrated the presence of a 42:58% mixture of α-pinene and β-pinene (FIGURE 4), the same product ratio previously described for the purified, native (-)-pinene synthase from Grand fir (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173). Chiral phase capillary GLC confirmed the products of the recombinant pinene synthase to be the (-)-(15^,:55)-enantiomers, as expected. No other monoteφene co-products were detected with the recombinant (-)-pinene synthase, as observed previously for the native enzyme (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293: 167-173).

Evidence for the formation of both α- and β-pinene by a single enzyme has been previously provided through co-purification studies, and differential inhibition and inactivation studies, as well as by isotopically sensitive branching experiments

(Gambliel, H., and Croteau, R. (1984) J. Biol. Chem. 259:740-748; Wagschal, K.C, Pyun, H.-J., Coates, R.M., and Croteau, R. (1994) Arch. Biochem. Biophys. 308:477- 99/02030

487; Wagschal, K., Savage, T.J., and Croteau, R. (1991) Tetrahedron 47:5933- 5944; Croteau, R., Wheeler, C.J., Cane, D.E., Ebert, R., and Ha, H.-J. (1987) Biochemistry 26:5383-5389). The cDNA cloning of pinene synthase provides the ultimate proof that a single enzyme forms both products. The calculated molecular weight of the (-)-pinene synthase deduced from AG3.18 (SEQ ID NO:3) is approximately 64,000 (excluding the putative transit peptide), which agrees well with the molecular weight of 63,000 established for the native enzyme from Grand fir by gel permeation chromatography and SDS-PAGE (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173). A limonene synthase cDNA has thus far been cloned only from two very closely related angiosperm species (Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024; Yuba, A., Yazaki, K., Tabata, M., Honda, G., and Croteau, R. (1996) Arch. Biochem. Biophys. 332:280-287), and the isolation of a pinene synthase cDNA has not been reported before. Pinene synthase has previously received considerable attention as a major defense-related monoterpene synthase in conifers (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289:267-273; Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173). In the Grand fir cDNA library, which was synthesized from mRNA obtained from wound- induced sapling stems, clones corresponding to pinene synthase are at least ten times more abundant than clones for myrcene synthase. This finding reflects the relative proportions of the induced levels of activities of these enzymes in Grand fir saplings; pinene synthase and limonene synthase are the major monoterpene synthase activities whereas the induced level of myrcene synthase activity is relatively low (Gijzen, M., Lewinsohn, E., and Croteau, R. (1991) Arch. Biochem. Biophys. 289:267-273). The cDNAs for inducible monoterpene synthases provide probes for genetic and molecular analysis of oleoresin-based defense in conifers. Northern blots (FIGURE 6) of total RNA extracted from non-wounded sapling stems and from stems two days after wounding (when enzyme activity first appears) were probed with cDNA fragments for AG2.2 (SEQ ID NOT), AG3.18 (SEQ ID NO:3) and AGIO (SEQ ID NO:5), and thus demonstrated that increased mRNA accumulation for monoterpene synthases is responsible for this induced, defensive response in Grand fir. The availability of cloned, defense-related monoterpene synthases presents several possible avenues for transgenic manipulation of oleoresin composition to improve tree resistance to bark beetles and other pests. For example, altering the 99/02030

monoterpene content of oleoresin may chemically disguise the host and decrease insect aggregation by changing the levels of pheromone precursors or predator attractants, or lower infestation by increasing toxicity toward beetles and their pathogenic fungal associates (Johnson, M.A., and Croteau, R. (1987) in Ecology and Metabolism of Plant Lipids (Fuller, G., and Nes, W.D., eds) pp. 76-91, American Chemical Society Symposium Series 325, Washington, DC; Gijzen, M., Lewinsohn, E., Savage, T.J., and Croteau, R.B. (1993) in Bioactive Volatile Compounds from Plants (Teranishi, R., Buttery, R.G., and Sugisawa, H., eds) pp. 8-22, American Chemical Society Symposium Series 525, Washington, DC; Byers, J.A. (1995) in Chemical Ecology of Insects 2 (Carde, R.T., and Bell, W.J., eds) pp. 154-213, Chapman and Hall, New York).

EXAMPLE 8

Properties of the Recombinant Monoterpene Synthases Encoded by cDNA Clones AG2.2 (SEQ ID NO:l),AG3.18 (SEQ ID NO:3) and AGIO (SEQ ID NO.S)

2+ . . 2+

All three recombinant enzymes require Mn for activity, and Mg is essentially ineffective as the divalent metal ion cofactor. This finding confirms earlier results obtained with the native monoterpene synthases of Grand fir and lodgepole pine (Pinus contorta) (Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173; Savage, T.J., Hatch, M.W., and Croteau, R. (1994) J. Biol. Chem. 269:4012-4020). All terpene synthases and prenyltransferases

2+ 2+ are thought to employ a divalent metal ion, usually Mg or Mn , in the ionization steps of the reaction sequence to neutralize the negative charge of the diphosphate leaving group (Croteau, R. (1987) Chem. Rev. 87:929-954; Cane, D.E. (1992) Ciba Found. Symp. Ser. 171:163-167; Poulter, CD., and Rilling, H.C. (1981) in Biosynthesis of Isoprenoid Compounds (Porter, J.W., and Spurgeon, S.L., eds) Vol. 1, pp. 161-224, Wiley & Sons, New York), and all relevant sequences thus far obtained bear a conserved aspartate rich element (DDXXD)(SEQ ID NO:45) considered to be involved in divalent metal ion binding (Stofer Vogel, B., Wildung, M.R., Vogel, G., and Croteau, R. (1996) J. Biol Chem. 271:23262-23268; Ashby, M.N., and Edwards, P.A. (1990) J. Biol. Chem. 265:13157-13164; Chen, A., Kroon, P.Λ., and Poulter, D.C. (1994) Protein Sci. 3:600-607; Tarshis, L.C, Yan, M., Poulter, CD., and Sacchettini, J.C. (1994) Biochemistry 33:10871-10877; Cane, D.E., Sohng, J.K., Lamberson, C.R., Rudnicki, S.M., Wu, Z., Lloyd, M.D., Oliver, J.S., and Hubbard, B.R. (1994) Biochemistry 33:5846-5857; Proctor, R.H., and 9/02030

Ho n, T.M. (1993) J. Biol. Chem. 268:4543-4548). In addition to this strict, general dependence on a divalent metal ion, the monoterpene synthases of conifers are unique in their further requirement for a monovalent cation (K ), a feature that distinguishes the gymnosperm monoterpene synthases from their counteφarts from angiosperm species and implies a fundamental structural and/or mechanistic difference between these two families of catalysts (Savage, T.J., Hatch, M.W., and Croteau, R. (1994) J. Biol. Chem. 269:4012-4020). All three recombinant monoterpene synthases depend upon K , with maximum activity achieved at approximately 500 M KC1. A requirement for K⁺ has been reported for a number of different types of enzymes, including those that catalyze phosphoryl cleavage or transfer reactions (Suelter, CH. (1970) Science 168:789-794) such as Hsc70 ATPase (Wilbanks, S.M., and McKay, D.B. (1995) J. Biol. Chem. 270:2251-2257). The

2+ + crystal structure of bovine Hsc70 ATPase indicates that both Mg and K interact directly with phosphate groups of the substrate and implicates three active site

2+ + aspartate residues in Mg and K binding (Wilbanks, S.M., and McKay, D.B. (1995) J. Biol. Chem. 270:2251-2257), reminiscent of the proposed role of the conserved DDXXD (SEQ ID NO:45) motif of the terpene synthases and prenyltransferases in divalent cation binding, a function also supported by recent site directed mutagenesis (Marrero, P.F., Poulter, CD., and Edwards, P.A. (1992) J. Biol. Chem. 267:21873- 21878; Joly, A., and Edwards, P.A. (1993) J. Biol. Chem. 268: 26983-26989; Song, L., and Poulter, CD. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:3044-3048; Koyama, T., Tajima, M., Sano, H., Doi, T., Koike-Takeshita, A., Obata, S., Nishino, T., and Ogura, K. (1996) Biochemistry 35:9533-9538) and by X-ray structural analysis (Tarshis, L.C, Yan, M., Poulter, CD., and Sacchettini, J.C (1994) Biochemistry 33:10871-10877) of farnesyl diphosphate synthase. cDNA cloning and functional expression of the myrcene, limonene and pinene synthases from Grand fir represent the first example of the isolation of multiple synthase genes from the same species, and provide tools for evaluation of structure-function relationships in the construction of acyclic, monocyclic and bicyclic monoterpene products and for detailed comparison to catalysts from phylogenetically distant plants that carry out ostensibly identical reactions (Gambliel, IT, and Croteau, R. (1984) J. Biol Chem. 259:740-748; Rajaonarivony, J.I.M., Gershenzon, J., and Croteau, R. (1992) Arch. Biochem. Biophys. 296:49-57; Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J., and Croteau, R. (1993) J Biol. Chem. 268:23016-23024; Adam, K.-P., Crock, J., and Croteau, R. (1996) Arch. 9/02030

Biochem. Biophys. 332:352-356). The recent acquisition of cDNA isolates encoding sesquiterpene synthases and diterpene synthases (Stofer Vogel, B., Wildung, M.R., Vogel, G., and Croteau, R. (1996) J. Biol Chem. 271:23262-23268) from Grand fir should, together with the monoterpene synthases, also permit addressing the structural basis of chain-length specificity for prenyl diphosphate substrates in this family of related enzymes.

EXAMPLE 9 Sequence Comparison of Cloned Monoterpene Synthases Previous studies based on substrate protection from inactivation with selective amino acid modifying reagents have implicated functionally important cysteine, histidine and arginine residues in a range of different monoterpene synthases (Rajaonarivony, J.I.M., Gershenzon, J., and Croteau, R. (1992) Arch. Biochem. Biophys. 296:49-57; Lewinsohn, E., Gijzen, M., and Croteau, R. (1992) Arch. Biochem. Biophys. 293:167-173; Savage, T.J., Hatch, M.W., and Croteau, R. (1994) J. Biol. Chem. 269:4012-4020; Rajaonarivony, J.I.M., Gershenzon, J., Miyazaki, J., and Croteau, R. (1992) Arch. Biochem. Biophys. 299:77-82; Savage, T.J., Ichii, H., Hume, S.D., Little, D.B., and Croteau, R. (1994) Arch. Biochem. Biophys. 320:257-265). Sequence alignment of 21 terpene synthases of plant origin (Colby, S.M., Alonso, W.R., Katahira, E.J., McGarvey, D.J., and Croteau, R. (1993) J. Biol. Chem. 268:23016-23024; Stofer Vogel, B., Wildung, M.R., Vogel, G., and Croteau, R. (1996) J. Biol. Chem. 271:23262-23268; Facchini, P.J., and Chappell, J. (1992) Proc. Natl. Acad. Sci. USA 89:11088-11092; Mau, C.J.D., and West, CA. (1994) Proc. Natl. Acad. Sci. USA 91:8497-8501; Yuba, A., Yazaki, K., Tabata, M.Honda, G., and Croteau, R. (1996) Arch. Biochem. Biophys. 332:280-287; Wildung, M.R., and Croteau, R. (1996) J. Biol. Chem. 271:9201-9204; Yamaguchi, S., Saito, T., Abe, H., Yamane, H., Murofushi, N., and Kamiya, Y. (1996) Plant J. 10:203-213; Dudareva, N., Cseke, L., Blanc, V.M., and Pichersky, E. (1996) Plant Cell 8:1137- 1148; Chen, X.-Y., Chen, Y., Heinstein, P., and Davisson, V.J. (1995) Arch. Biochem. Biophys. 324:255-266; Chen, X.-Y., Wang, M., Chen, Y., Davisson, J., and Heinstein, P. (1996) J. Nat. Prod. 59:944-951; Back, K., and Chappell, J. (1995) J. Biol. Chem. 270:7375-7381) reveals two absolutely conserved arginine residues, corresponding to Arg and Arg of pinene synthase (SEQ ID ΝO:4), one highly

543 conserved cysteine residue (pinene synthase Cys )(SEQ ID NO:4), and one highly conserved histidine residue (pinene synthase His )(SEQ ID NO:4). The DDXXD

379 380 383 (SEQ ID NO:45) sequence motif (pinene synthase Asp , Asp and Asp ) (SEQ 99/02030

ID NO:4) is absolutely conserved in all relevant plant terpene synthases, as are

198 248 322 329 several other amino acid residues corresponding to Phe , Leu , Glu , Trp , Trp ' and Pro of pinene synthase (SEQ ID NO:4).

Amino acid sequences of the plant terpene synthases were compared with each other and with the deduced sequences of several sesquiterpene synthases cloned from microorganisms (Proctor, R.H., and Hohn, T.M. (1993) J. Biol. Chem. 268:4543-4548; Back, K., and Chappell, J. (1995) J. Biol. Chem. 270:7375- 7381 ; Hohn, T.M., and Desjardins, A.E. (1992) Mol. Plant-Microbe Interactions 5:249-256). As with all other plant terpene synthases, no significant conservation in primary sequence exists between the monoterpene synthases from Grand fir and the terpene synthases of microbial origin, except for the DDXXD (SEQ ID N :45) sequence motif previously identified as a common element of all terpene synthases, and prenyltransferases which employ a related electrophilic reaction mechanism (Croteau, R., Wheeler, C.J., Cane, D.E., Ebert, R., and Ha, H.-J. (1987) Biochemistry 26:5383-5389; Chen, A., Kroon, P.A., and Poulter, D.C (1994) Protein Sci. 3:600-607; McCaskill, D., and Croteau, R. (1997) Adv. Biochem. Engineering Biotech. 55:108-146). The evidence is presently insufficient to determine whether extant plant and microbial terpene synthases represent divergent evolution from a common ancestor, which may also have given rise to the prenyltransferases, or whether these similar catalysts evolved convergently.

EXAMPLE 10 Alteration of Monoterpene Levels and Composition in Plant Seeds In accordance with the present invention, methods for increasing production of monoterpene compounds in a plant, particularly in plant seeds, are provided. The methods involve transforming a plant cell with a nucleic acid sequence encoding at least one gymnosperm monoterpene synthase, such as those encoded by the nucleic acid sequences set forth in SEQ ID NOT, SEQ ID NO:3 and SEQ ID NO:5. This has the effect of altering monoteφene biosynthesis, thereby increasing the production of monoterpenes, as well as providing novel seed oils having desirable monoterpene compositions. In this manner, the transformed seed provides a factory for the production of modified oils. The modified oil itself may be used and/or the compounds in the oils can be isolated. Thus, the present invention allows for the production of particular monoterpenes of interest as well as speciality oils.

The nucleic acid encoding the monoterpene synthases of the present invention can be used in expression cassettes for expression in the transformed plant tissues. To alter the monoterpene levels in a plant of interest, the plant is transformed with at least one expression cassette comprising a transcriptional initiation region linked to a nucleic acid sequence encoding a monoterpene synthase. Such an expression cassette is provided with a plurality of restriction sites for insertion of the nucleic acid sequence encoding a monoterpene synthase so that it is under the transcriptional regulation of the regulatory regions.

The transcriptional initiation sequence may be native or analogous to the host or foreign or heterologous to the host. In this regard, the term "foreign" means that the transcriptional initiation sequence is not found in the wild-type host into which the transcriptional initiation region is introduced.

Of particular interest are those transcriptional initiation regions associated with storage proteins, such as napin, cruciferin, β-conglycinin, phaseolin, globulin or the like, and proteins involved in fatty acid biosynthesis, such as acyl carrier protein (ACP). See, U.S. Patent No. 5,420,034, herein incorporated by reference. The transcriptional cassette will preferably include, in the 5' to 3' direction of transcription, a transcriptional and translational initiation region, a gymnosperm monoterpene synthase DNA sequence of interest, and a transcriptional and translational termination region functional in plants. The termination region may be from the same organism as the transcriptional initiation region, may be from the same organism as the monoterpene synthase DNA, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. Other termination sequences are set forth in Guerineau et al., (1991), Mol. Gen. Genet., 262:141-144; Proudfoot, (1991), Cell, 64:671-674; Sanfacon et al., (1991), Genes Dev., 5:141-149; Mogen et al., (1990), Plant Cell, 2:1261-1272; Munroe et al., (1990), Gene, 91:151-15 8; Ballas et al., (1989), Nucleic Acids Res., 17:7891-7903; Joshi et al., (1987), Nucleic Acid Res., 15:9627-9639).

In the presently preferred form of the invention, a nucleic acid sequence encoding a gymnosperm monoterpene synthase protein will be targeted to plastids, such as chloroplasts, for expression. Thus, the nucleic acid sequence, or sequences, encoding a gymnosperm monoterpene synthase protein, or proteins, may be inserted into the plastid for expression with appropriate plastid constructs and regulatory elements. Alternatively, nuclear transformation may be used in which case the expression cassette will contain a nucleic acid sequence encoding a transit peptide to direct the monoterpene biosynthesis enzyme of interest to the plastid. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; della- Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and, Shah et al. (1986) Science 233:478- 481. Nucleic acid sequences encoding gymnosperm monoteφene synthases of the present invention may utilize native or heterologous transit peptides.

The construct may also include any other necessary regulators such as plant translational consensus sequences (Joshi, C.P., (1987), Nucleic Acids Research,

15:6643-6653), introns (Luehrsen and Walbot, (1991), Mol. Gen. Genet., 225:81-93) and the like, operably linked to a nucleotide sequence encoding a monoteφene synthase of the present invention.

It may be beneficial to include 5' leader sequences in the expression cassette which can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, O., Fuerst, T.R., and Moss, B. (1989) PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology, 154:9- 20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D.G., and Sarnow, P. (1991), Nature, 353:90-94; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S.A., and Gehrke, L. (1987), Nature, 325:622-625; tobacco mosaic virus leader (TMV), (Gallie, D.R. et al. (1989), Molecular Biology of RNA, pages 237-256; and maize chlorotic mottle virus leader (MCMV) (Lommel, S.A. et al. (1991), Virology, 81:382-385. See also, Della-Cioppa et al., (1987), Plant Physiology, 84:965-968. Depending upon where the monoterpene synthase sequence of interest is to be expressed, it may be desirable to synthesize the sequence with plant preferred codons, or alternatively with chloroplast preferred codons. The plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in the largest amount in the particular plant species of interest. See, EPA 0359472; EPA 0385962; WO 91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) Nucleic Acids Research 17:477-498. In this manner, the nucleotide sequences can be optimized for expression in any plant. It is recognized that all or any part of the nucleic acid sequence encoding a gymnosperm monoterpene synthase protein may be optimized or synthetic. That is, synthetic or partially optimized sequences may also be used. For the construction of chloroplast preferred genes, see U.S. Patent No. 5,545,817.

In preparing the transcription cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and in the proper reading frame. Towards this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resection, ligatiυn, or the like may be employed, where insertions, deletions or substitutions, such as transitions and transversions, may be involved.

The recombinant DNA molecules of the invention can be introduced into the plant cell in a number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might depend on the type of plant, i.e., monocot or dicot, targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) BioTechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium mediated transformation (Hinchee et al. (1988) Biotechnology 6:915-921) and ballistic particle acceleration (see, for example, Sanford et al., U.S. Patent No. 4,945,050; and McCabe et al. (1988) Biotechnology 6:923-926). Also see, Weissinger et al. (1988) Annual Rev. Genet. 22:421-477; Sanford et al. (1987) Paniculate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al (1988) Proc. Natl. Acad Sci. USA, 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fronun et al. (1990) Biotechnology 8:833-839; and Gordon-Kamm et al. (1990) Plant Cell 2:603-618 (maize).

Alternatively, a plant plastid can be transformed directly. Stable transformation of chloroplasts has been reported in higher plants, see, for example, SVAB et al. (1990) Proc. Nat'l. Acad. Sci. USA 87:85268530; SVAB & Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Staub & Maliga (1993) Embo J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. In such methods, plastid gene expression can be accomplished by use of a plastid gene promoter or by trans-activation of a silent plastid-borne transgene positioned for expression from a selective promoter sequence such as that recognized by T7 RNA polymerase. The silent plastid gene is activated by expression of the specific RNA polymerase from a nuclear expression construct and targeting of the polymerase to the plastid by use of a transit peptide. Tissue- specific expression may be obtained in such a method by use of a nuclear-encoded and plastid-directed specific RNA polymerase expressed from a suitable plant tissue specific promoter. Such a system has been reported in McBride et al. (1994) Proc. Nail. AcadSci. USA 91:7301-7305.

The cells which have been transformed may be grown into plants by a variety of art-recognized means. See, for example, McConnick et al., Plant Cell Reports (1986), 5:81-84. These plants may then be grown, and either selfed or crossed with a different plant strain, and the resulting homozygotes or hybrids having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure the desired phenotype or other property has been achieved.

As a host cell, any plant variety may be employed. Of particular interest, are plant species which provide seeds of commercial value. For the most part, plants will be chosen where the seed is produced in high amounts, a seed-specific product of interest is involved, or the seed or a seed part is edible. Seeds of interest in the practice of the present invention include, but are not limited to, the oil seeds, such as oilseed Brassica seeds, cotton seeds, soybean, safflower, sunflower, coconut, palm, and the like; grain seeds such as wheat, barley, oats, amaranth, flax, rye, triticale, rice and corn; other edible seeds or seeds with edible parts including pumpkin, squash, sesame, poppy, grape, mung beans, peanut, peas, beans, radish, alfalfa, cocoa, and coffee; and tree nuts such as walnuts, almonds, pecans, and chick-peas.

EXAMPLE 11 A Strategy For Cloning Gymnosperm Monoterpene Synthases The present invention includes gymnosperm monoterpene synthase proteins, and nucleic acid molecules that encode gymnosperm monoterpene synthase proteins. The amino acid sequence of each of the gymnosperm monoteφene synthase proteins of the present invention each includes al least one of the amino acid sequence elements disclosed in Table 1.

Table 1

The numbers set forth in Table 1 for the first and last amino acid residue of each of the peptide sequences is the number of the corresponding amino acid residue in the amino acid sequence of the (-)-pinene synthase (SEQ ID NO:4) isolated from Abies grandis. Where a sequence of amino acid residues appears in brackets, e.g., (L,IN) in Table 1, the first amino acid residue within the brackets is the residue that appears in the (-)-pinene synthase amino acid sequence set forth in SEQ ID ΝO:4. The subsequent amino acid residues within the brackets represent other amino acid residues that commonly occur at the corresponding position in the amino acid sequence of other Abies grandis enzymes involved in teφene synthesis.

In Table 1, the letter "F" refers to the forward PCR reaction, i.e., the PCR reaction which synthesizes the sense nucleic acid strand that encodes a gymnosperm monoteφene synthase. The letter "R" refers to the reverse PCR reaction, i.e., the PCR reaction that synthesizes the antisense nucleic acid molecule that is complementary to the sense nucleic acid strand synthesized in the forward PCR reaction. In order to clone nucleic acid molecules encoding gymnosperm monoteφene synthases of the present invention, one or more oligonucleotide molecules corresponding to at least a portion of one of the amino acid sequences set forth in Table 1 can be used as a probe or probes with which to screen a genomic or cDNA library derived from one or more gymnosperm species. In this context, the term "corresponding," or "correspond" or "corresponds," means that the oligonucleotide base sequence either a) encodes all or part of at least one of the amino acid sequences set forth in Table 1, or b) is complementary to a base sequence that encodes all or part of at least one of the amino acid sequences set forth in Table 1. The oligonucleotide probe(s) may contain a synthetic base, such as inosine, which can be substituted for one or more of the four, naturally-occurring bases, i.e., adenine ("A"), guanine ("G"), cytosine ("C") and thymine ("T"). Thus, for example, the following oligonucleotide sequences "correspond" to the tripeptide sequence M M M: 5ΑTGATGATG3' (sense orientation) (SEQ ID NO:54); 3 ACTACTAC5' (antisense orientation) (SEQ ID NO:55) and 3'IACIACIAC5' (SEQ ID NO:56).

One or more oligonucleotide sequence(s), corresponding to at least a portion of at least one of the amino acid sequences set forth in Table 1 , can be used to screen a nucleic acid library in order to identify monoterpene synthase clones of the present invention, according to methods well known to one of ordinary skill in the art. See, e.g., Sambrook et al, supra. The stringency of the hybridization and wash conditions during library screening in accordance with the present invention, utilizing one or more oligonucleotide sequence(s) corresponding to at least a portion of at least one of the amino acid sequences set forth in Table 1 , is at least: for the hybridization step, 6X SSPE, 40-45°C, for 36 hours; for the wash step, 3X SSPE, 45°C, 3 X 15 minute washes. The presently preferred hybridization and wash conditions during library screening, utilizing one or more oligonucleotide sequence(s) corresponding to at least a portion of at least one of the amino acid sequences set forth in Table 1, in accordance with the present invention are: for the hybridization step, 6X SSPE, 40- 45°C, for 36 hours; for the wash step, 0.1X SSPE, 65°C-70°C, 3 X 15 minute washes. Examples of oligonucleotide sequences, corresponding to at least one of the amino acid sequences set forth in Table 1, that hybridize, under the foregoing hybridization and wash conditions, to the sense strands of the nucleic acid sequences of the present invention that encode gymnosperm monoteφene synthase proteins are set forth in Table 2. Table 2

Similarly, each of the gymnosperm monoteφene synthase clones set forth in SEQ ID NOT, SEQ ID NO: 3 and SEQ ID NO: 5, or a portion thereof, may be used as a probe to screen a nucleic acid library in order to isolate monoteφene synthase clones of the present invention, according to methods well known to one of ordinary skill in the art. See, e.g., Sambrook et al, supra. The stringency of the hybridization and wash conditions during library screening in accordance with the present invention is at least: for the hybridization step, 6X SSPE buffer at 45°C to 50°C for 36 hours; for the wash step, 3X SSPE buffer at 50°C (three, fifteen minute washes). In accordance with the present invention, the presently preferred hybridization and wash conditions during library screening utilizing any of the gymnosperm monoteφene synthase clones set forth in SEQ ID NOT, SEQ ID NO:3 and SEQ ID NO: 5, or a portion thereof, as probe are: for the hybridization step, 6X SSPE, 40- 45°C, for 36 hours; for the wash step, 0.1X SSPE, 70°C-75°C, 3 X 15 minute washes. Additionally, at least two oligonucleotide sequence(s), each corresponding to at least a portion of at least one of the amino acid sequences set forth in Table 1 , can be used in a PCR reaction to generate a portion of a monoteφene synthase clone of the present invention, which can be used as a probe to isolate a full-length clone of a monoteφene synthase clone of the present invention. Thus, oligonucleotides that are useful as probes in the forward PCR reaction correspond to at least a portion of at least one of the amino acid sequences disclosed in Table 1 as having the "F" orientation. Conversely, oligonucleotides that are useful as probes in the reverse PCR reaction correspond to at least a portion of at least one of the amino acid sequences disclosed in Table 1 as having the "R" orientation. PCR reactions can be carried out according to art-recognized PCR reaction conditions, such as the PCR reaction conditions set forth in Example 1 herein and as set forth in "PCR Strategies", M.A. Innis, D.H. Gelfand and J.J. Sninsky, eds., 1995, Academic Press, San Diego, CA (Chapter 14); "PCR Protocols: A Guide to Methods and Applications", M.A. Innis, D.H. Gelfand, J.J. Sninsky and TJ. White, eds., Academic Press, NY (1990). The presently preferred PCR reaction conditions are:

dNTPs 200 μM each

MgCl₂ 5-7 mM

F and R primers 100 nM - 1 μM each

Taq polymerase 1-2 units/reaction cDNA template 10-100 ng/reaction

Buffers, PCR grade water, and Chill-out wax or mineral oil

The presently preferred thermocycler conditions are:

Denaturation 94° x 2 min 1 cycle

Denaturation 94° x 45 s 35 cycles

Annealing 42° - 55° x 45 s - 1 min

Polymerization 72 x 1 -2 min

Adenylation 72 x 10 min 1 cycle

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An isolated nucleotide sequence encoding a gymnosperm monoteφene synthase, said isolated nucleotide sequence being capable of hybridizing under stringent conditions to at least one oligonucleotide having a base sequence that corresponds to at least one peptide sequence selected from the group consisting of SEQ ID NO:46. SEQ ID NO:47. SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

2. An isolated nucleotide sequence of Claim 1 encoding a gymnosperm myrcene synthase.

3. An isolated nucleotide sequence of Claim 1 encoding a gymnosperm limonene synthase.

4. An isolated nucleotide sequence of Claim 1 encoding a gymnosperm pinene synthase.

5. An isolated nucleotide sequence of Claim 1 encoding a Grand fir (Abies grandis) monoteφene synthase.

6. An isolated nucleotide sequence of Claim 1 encoding a Grand fir (Abies grandis) myrcene synthase.

7. An isolated nucleotide sequence of Claim 1 encoding a Grand fir (Abies grandis) limonene synthase.

8. An isolated nucleotide sequence of Claim 1 encoding a Grand fir (Abies grandis) pinene synthase.

9. An isolated nucleotide sequence of Claim 1 having the sequence of SEQ ID NOT .

10. An isolated nucleotide sequence of Claim 1 having the sequence of SEQ ID NO:3.

11. An isolated nucleotide sequence of Claim 1 having the sequence of SEQ ID NO:5

12. An isolated nucleotide sequence of Claim 1 which encodes the amino acid sequence of SEQ ID NO:2.

13. An isolated nucleotide sequence of Claim 1 which encodes the amino acid sequence of SEQ ID NO:4

14. An isolated nucleotide sequence of Claim 1 which encodes the amino acid sequence of SEQ ID NO:6.

15. An isolated nucleotide sequence encoding a gymnosperm monoteφene synthase protein, said isolated nucleotide sequence having a complementary nucleotide sequence that is capable of hybridizing to the nucleotide sequence of any one of SEQ ID NOT. SEQ ID NO:3 and SEQ ID NO:5.

16. An isolated nucleic acid molecule encoding a gymnosperm monoteφene synthase, said nucleic acid molecule being capable of hybridizing to at least two oligonucleotides, each of said oligonucleotides having a base sequence that corresponds to at least one peptide sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

17. An isolated nucleic acid molecule encoding a Grand fir (Abies grandis) monoteφene synthase, said nucleic acid molecule being capable of hybridizing to at least two oligonucleotides, each of said oligonucleotides having a base sequence that corresponds to at least one peptide sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

18. An isolated gymnosperm monoteφene synthase protein, said gymnosperm monoteφene synthase protein including at least one amino acid sequence element selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

19. An isolated gymnosperm myrcene synthase protein, said gymnosperm myrcene synthase protein including at least one amino acid sequence element selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

20. An isolated gymnosperm pinene synthase protein, said gymnosperm pinene synthase protein including at least one amino acid sequence element selected from the group consisting of SEQ ID NO:46. SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

21. An isolated gymnosperm limonene synthase protein, said gymnosperm limonene synthase protein including at least one amino acid sequence element selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

22. An isolated Grand fir (Abies grandis) monoteφene synthase protein, said Grand fir (Abies grandis) monoteφene synthase protein including at least one amino acid sequence element selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

23. An isolated Grand fir (Abies grandis) monoteφene synthase protein of Claim 22, said monoteφene synthase protein having the sequence of SEQ ID NO:2.

24. An isolated Grand fir (Abies grandis) monoteφene synthase protein of Claim 22, said monoteφene synthase protein having the sequence of SEQ ID NO:4.

25. An isolated Grand fir (Abies grandis) monoteφene synthase protein of Claim 22, said monoteφene synthase protein having the sequence of SEQ ID NO:6.

26. A replicable expression vector comprising a nucleotide sequence encoding a gymnosperm monoteφene synthase, said nucleotide sequence encoding a gymnosperm monoteφene synthase being capable of hybridizing at high stringency to at least one oligonucleotide having a base sequence that corresponds to at least one peptide sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

27. A replicable expression vector of Claim 26 wherein said nucleotide sequence encoding a gymnosperm monoteφene synthase encodes a Grand fir (Abies grandis) monoteφene synthase.

28. A host cell comprising a vector of any one of claims 26 and 27.

29. A replicable expression vector comprising a nucleotide sequence encoding a gymnosperm monoteφene synthase, said nucleotide sequence having a complementary nucleotide sequence that is capable of hybridizing to the nucleotide sequence of any one of SEQ ID NOT. SEQ ID NO:3 and SEQ ID NO:5.

30. A host cell comprising a vector of Claim 29.

31. A method of enhancing the production of a gymnosperm monoteφene synthase in a suitable host cell comprising introducing into the host cell an expression vector of Claim 26 under conditions enabling expression of the gymnosperm monoteφene synthase in the host cell.

32. A method for enhancing monoteφene levels in a host cell, said method comprising transforming said host cell with a nucleic acid construct comprising at least one nucleic acid sequence defining a transcriptional initiation region from a gene expressed in the host cell and at least one nucleic acid sequence encoding a gymnosperm monoteφene synthase, said nucleic acid sequence encoding a gymnosperm monoteφene synthase being capable of hybridizing at high stringency to at least one oligonucleotide having a base sequence that corresponds to at least one peptide sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

33. The method of Claim 32 wherein said nucleic acid sequence encoding a gymnosperm monoteφene synthase encodes a Grand fir (Abies grandis) monoteφene synthase.

34. A method for altering monoteφene levels in a seed from a host plant, said method comprising transforming said host plant with a nucleic acid construct comprising at least one nucleic acid sequence defining a transcriptional initiation region from a gene expressed in a plant seed and at least one nucleic acid sequence encoding a gymnosperm monoteφene synthase, said nucleic acid sequence encoding a gymnosperm monoteφene synthase being capable of hybridizing at high stringency to at least one oligonucleotide having a base sequence that corresponds to at least one peptide sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

35. The method of Claim 34 wherein the transcriptional initiation region is from a gene preferentially expressed in a plant seed.

36. The method of Claim 34 wherein said nucleic acid sequence encoding a gymnosperm monoteφene synthase encodes a Grand fir (Abies grandis) monoteφene synthase.

37. The method of Claim 34 wherein said gymnosperm monoteφene synthase is not native to said host plant.

38. A transgenic plant produced according to the method of Claim 34.

39. A method for increasing the monoteφene biosynthetic flux in seed from a host plant, said method comprising transforming said host plant with a construct comprising as operably linked components, a transcriptional initiation region from a gene preferentially expressed in a plant seed, a plastid transit peptide, a DNA sequence encoding a gymnosperm monoteφene synthase protein, and a transcriptional termination region, said DNA sequence encoding a gymnosperm monoteφene synthase protein being capable of hybridizing at high stringency to at least one oligonucleotide having a base sequence that corresponds to at least one peptide sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

40. The method of Claim 39, wherein said DNA sequence encoding a gymnosperm monoteφene synthase protein encodes an enzyme selected from the group consisting of limonene synthase, myrcene synthase and pinene synthase.

41. A method for producing a monoteφene compound in a seed, said method comprising the steps of obtaining a transformed plant which produces said seed, said plant having and expressing in its genome:

a nucleic acid sequence encoding a gymnosperm monoteφene synthase protein which is operably linked to a plastid transit peptide and a transcriptional initiation region from a gene preferentially expressed in a plant seed, wherein said nucleic acid sequence encoding a gymnosperm monoteφene synthase protein is capable of hybridizing at high stringency to at least one oligonucleotide having a base sequence that corresponds to at least one peptide sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49. SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53.

42. A seed transformed with a nucleic acid sequence encoding a gymnosperm monoteφene synthase protein, said nucleic acid sequence encoding a gymnosperm monoteφene synthase protein being capable of hybridizing at high stringency to at least one oligonucleotide having a base sequence that corresponds to at least one peptide sequence selected from the group consisting of SEQ ID NO:SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52 and SEQ ID NO:53, said transformed seed having altered monoteφene levels.

43. The transformed seed of claim 42, wherein said seed produces increased levels of at least one monoteφene compound, said monoteφene compound selected from the group consisting of (-)-4S-limonene, (-)-α-pinene. myrcene, 4S-β- phellandrene.

44. The transformed seed of claim 43, wherein said seed produces increased levels of (-)4S-limonene.

45. Seed produced by the method of any one of claims 34, 35, 36, 37, 39, 40 and 41.

46. Plants produced by the method of any one of claims 34, 35, 36, 37, 39, 40 and 41.

47. Oil extracted from seeds produced by the method of any one of claims 34, 35, 36, 37, 39, 40 and 41.

48. Meal extracted from seed produced by the method of any one of claims 34, 35, 36, 37, 39, 40 and 41.

49. The method of any one of claims 34, 35, 36, 37, 39, 40 and 41, wherein said seed is from a plant selected from the group consisting of oilseed Brassica, cotton, soybean, safflower, sunflower, coconut, palm, wheat, barley, rice, corn, oats, amaranth, pumpkin, squash, sesame, poppy, grape, mung beans, peanut, peas, beans, radish, alfalfa, cocoa, coffee, and tree nuts.

50. The method of Claim 49, wherein said seed is from an oilseed crop plant selected from the group consisting of oilseed brassica, cotton, soybean, safflower, sunflower, palm, coconut, and corn.